Sequential reactions of surface- tethered glycolytic enzymes.
ABSTRACT The development of complex hybrid organic-inorganic devices faces several challenges, including how they can generate energy. Cells face similar challenges regarding local energy production. Mammalian sperm solve this problem by generating ATP down the flagellar principal piece by means of glycolytic enzymes, several of which are tethered to a cytoskeletal support via germ-cell-specific targeting domains. Inspired by this design, we have produced recombinant hexokinase type 1 and glucose-6-phosphate isomerase capable of oriented immobilization on a nickel-nitrilotriacetic acid modified surface. Specific activities of enzymes tethered via this strategy were substantially higher than when randomly adsorbed. Furthermore, these enzymes showed sequential activities when tethered onto the same surface. This is the first demonstration of surface-tethered pathway components showing sequential enzymatic activities, and it provides a first step toward reconstitution of glycolysis on engineered hybrid devices.
- SourceAvailable from: Chinatsu Mukai[Show abstract] [Hide abstract]
ABSTRACT: Maintaining activity of enzymes tethered to solid interfaces remains a major challenge in developing hybrid organic-inorganic devices. In nature, mammalian spermatozoa have overcome this design challenge by having glycolytic enzymes with specialized targeting domains that enable them to function while tethered to a cytoskeletal element. As a step toward designing a hybrid organic-inorganic ATP-generating system, we implemented a biomimetic site-specific immobilization strategy to tether two glycolytic enzymes representing different functional enzyme families: triose phosphoisomerase (TPI; an isomerase) and glyceraldehyde 3-phosphate dehydrogenase (GAPDHS; an oxidoreductase). We then evaluated the activities of these enzymes in comparison to when they were tethered via classical carboxyl-amine crosslinking. Both enzymes show similar surface binding regardless of immobilization method. Remarkably, specific activities for both enzymes were significantly higher when tethered using the biomimetic, site-specific immobilization approach. Using this biomimetic approach, we tethered both enzymes to a single surface and demonstrated their function in series in both forward and reverse directions. Again, the activities in series were significantly higher in both directions when the enzymes were coupled using this biomimetic approach versus carboxyl-amine binding. Our results suggest that biomimetic, site-specific immobilization can provide important functional advantages over chemically specific, but non-oriented attachment, an important strategic insight given the growing interest in recapitulating entire biological pathways on hybrid organic-inorganic devices.PLoS ONE 01/2013; 8(4):e61434. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Sperm utilize glycolysis to generate ATP required for motility and several spermatogenic cell-specific glycolytic isozymes are associated with the fibrous sheath (FS) in the principle piece of the sperm flagellum. We used proteomics and molecular biology approaches to confirm earlier reports that a novel enolase is present in mouse sperm. We then found that a pan-enolase antibody, but not antibodies to ENO2 and ENO3, recognized a protein in the principal piece of the mouse sperm flagellum. Database analyses identified two previously uncharacterized enolase family-like candidate genes, 64306537H0Rik and Gm5506. Northern analysis indicated that 64306537H0Rik (renamed Eno4) was transcribed in testes of mice by Postnatal Day 12. To determine the role of ENO4, we generated mice using ES cells in which an Eno4 allele was disrupted by a gene trap containing a beta galactosidase (beta-gal) reporter (Eno4+/Gt). Expression of beta-gal occurred in the testis and male mice homozygous for the gene trap allele (Eno4Gt/Gt) were infertile. Epididymal sperm numbers were two-fold lower and sperm motility was reduced substantially in Eno4Gt/Gt mice compared to wild type (WT) mice. Sperm from Eno4Gt/Gt mice had a coiled flagellum and a disorganized FS. The Gm5506 gene encodes a protein identical to ENO1 and also is transcribed at a low level in testis. We conclude that ENO4 is required for normal assembly of the FS and provides most of the enolase activity in sperm, and that Eno1 and/or Gm5506 may encode a minor portion of the enolase activity in sperm.Biology of Reproduction 02/2013; · 4.03 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Scaffold proteins are involved in many enzyme cascades in signaling pathways and metabolic processes. The study of scaffolds occurring in biological systems advances at a rapid pace and recently developed engineered synthetic scaffolds enable the precise placement of components of an enzymatic cascade within nanometer distances. Recent experimental results demonstrate significantly increased throughput of enzymatic cascades as a result of the utilization of a scaffold, but our understanding of the mechanisms responsible for this increase is not complete. We discuss the physics of diffusive transport processes relevant for these unique reaction-diffusion systems.Current opinion in biotechnology 01/2013; · 7.82 Impact Factor
Chemistry & Biology
Sequential Reactions of Surface-
Tethered Glycolytic Enzymes
Chinatsu Mukai,1Magnus Bergkvist,2,3Jacquelyn L. Nelson,1and Alexander J. Travis1,*
1Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
2Nanobiotechnology Center, Cornell University, Ithaca, NY 14853, USA
3Present address: College of Nanoscale Science and Engineering, University at Albany, State University of New York, 255 Fuller Rd,
Albany, NY 12203, USA
The development of complex hybrid organic-inor-
ganic devices faces several challenges, including
how they can generate energy. Cells face similar
Mammalian sperm solve this problem by generating
ATP down the flagellar principal piece by means of
glycolytic enzymes, several of which are tethered
to a cytoskeletal support via germ-cell-specific tar-
geting domains. Inspired by this design, we have
produced recombinant hexokinase type 1 and
glucose-6-phosphate isomerase capable of oriented
immobilization on a nickel-nitrilotriacetic acid modi-
fied surface. Specific activities of enzymes tethered
via this strategy were substantially higher than
when randomly adsorbed. Furthermore, these en-
zymes showed sequential activities when tethered
onto the same surface.This isthe first demonstration
of surface-tethered pathway components showing
sequential enzymatic activities, and it provides a first
step toward reconstitution of glycolysis on engi-
neered hybrid devices.
Hybrid organic-inorganic devices derive the potential benefits of
combining biological functions with nonbiological material
systems. However, their practical development is faced with
that is desired for many devices is the ability to generate energy
from available substrates.
Because ATP supplies the power for many biological reac-
tions, much attention has been focused on devices or systems
that can produce their own ATP. Two systems to generate
ATP have been reported that utilize the F0F1-ATP synthase
(Luo et al., 2005; Steinberg-Yfrach et al., 1998). An alternative
approach has been described that uses pyruvate kinase to
generate ATP, which was used to power kinesin molecules (Du
et al., 2005). These reports demonstrated not only that it is
possible to engineer devices that can make ATP, but also that
the ATP produced could be used to power desired molecular
outputs. However, the F0F1-ATP synthase systems require
exogenous light (photons), which is not practical for future
in vivo medical applications. Similarly, the pyruvate kinase
system relies on administration of phosphoenolpyruvate, which
is also not freely available outside of cells. Thus, there are limita-
tions when trying to translate these approaches into practical
that must function using substrates available in an organism.
Regardless of the end goals of such devices, a technical
obstacle common to the field is how to retain the activity of bio-
logical macromolecules while they are attached to a solid
appropriate to engage with a substrate; they also must have
access to that substrate, and when substrate is bound, many
enzymes must change orientation or conformation in some
fashion to carry out a reaction. Introducing specific domains or
amino acid sequences in recombinant proteins is one strategy
to tether them to a surface in an orderly fashion; however, the
position and nature of such introduced sequences can greatly
influence that protein’s molecular function (Fischer and Hess,
as part of chemical synthetic processes (Schoemaker et al.,
2003). Difficulties associated with enzyme function on a solid
tion substrates to solid supports (Basso et al., 2006; Laurent
et al., 2008). However, this strategy is not suitable for technical
applications such as those discussed above, because the
enzymes would not naturally be found in the device’s environ-
ment, unlike metabolizable substrates such as glucose.
In this article, we seek to address the technical issue of main-
taining protein function while bound to an inorganic support, in
the context of reconstituting the first sequential steps in the
metabolic pathway of glycolysis. This pathway converts glucose
into pyruvate, releasing energy that can be used to form ATP.
Biology can provide inspirations for solutions to these technical
and functional challenges (Ummat et al., 2005). For example, our
work on mammalian sperm, along with that from other labs, has
revealed how these cells have evolved variants of glycolytic
enzymes that are bound to a solid, cytoskeletal structure known
as the ‘‘fibrous sheath’’ (Cao et al., 2006; Krisfalusi et al., 2006;
Storey and Kayne, 1975; Travis and Kopf, 2002; Westhoff and
Kamp, 1997). These enzymes differ from their somatic counter-
parts in having domains that are believed to be involved in
protein targeting to the fibrous sheath (Mori et al., 1998; Travis
et al., 1998; Travis et al., 1999; Welch et al., 1992). This structure
Chemistry & Biology 16, 1013–1020, September 25, 2009 ª2009 Elsevier Ltd All rights reserved 1013
is found only in the flagellar principal piece, which is distal to the
midpiece—the only region of sperm to contain mitochondria
(Figure 1). Thus, glycolysis can produce ATP locally where it is
needed to power flagellar motility and the regulators of that
motility (Miki et al., 2004; Mukai and Okuno, 2004; Travis et al.,
The almost ‘‘solid-state’’ arrangement of glycolytic enzymes
strategy for oriented immobilization of these enzymes on inor-
ganic supports. Namely, we hypothesized that replacement of
to an inorganic support would result in an oriented immobiliza-
tion that left the protein functional. This strategy therefore seeks
to reduce the trial and error normally associated with this goal.
Using this biomimetic approach, this article describes the
coupling and sequential activities of the first two enzymes of
glycolysis when immobilized together on a single, solid support.
Toward this goal, we generated recombinant forms of the male
germ-cell-specific hexokinase (HK: NM 010438; catalyzes the
conversion of glucose to glucose-6-phosphate, utilizing ATP),
and glucose-6-phosphate isomerase (GPI: NM 008155; cata-
lyzes the conversion of glucose-6-phosphate to fructose-6-
phosphate), and then tested their functions individually and in
series on the same solid support. To our knowledge, we believe
this to be the first demonstration of tethered pathway compo-
nents showing sequential enzymatic activities, moving one
step toward future hybrid organic-inorganic devices.
Generation of Recombinant HK1 and GPI
The complementary deoxyribonucleic acid (cDNA) of a germ-
cell-specific isoform of HK1 (HK1-sc), and GPI were obtained
by reverse-transcription polymerase chain reaction (RT-PCR)
from mouse testis RNA, and the germ-cell-specific targeting
domain of HK (Travis et al., 1999) was removed by nested
PCR. The constructs were inserted into a vector that places a
hexahistidine (His-) tag at the amino terminus, thus replacing
the HK1-sc germ-cell-specific targeting domain. His-HK and
His-GPI were expressed in a mammalian expression system
and then purified using a nickel-nitrilotriacetic acid (Ni-NTA)
system. The products were separated using SDS-PAGE, and
theirpurityandidentity wereverified throughtheuseofCoomas-
sie staining and immunoblotting with antibodies against the
respective proteins and against the His-tag. Both recombinant
enzymes were found to be highly pure (80%–90%, with only
slight variations between sample preparations), immunoreactive
with the appropriate antibodies, and to migrate at the expected
molecular weights (Figure 2).
Quantification of His-HK and His-GPI Enzyme Activities
We examined the activity of the recombinant proteins individu-
ally in solution by means of a coupled reaction pathway,
ultimately measuring the reduction of nicotinamide adenine
dinucleotide phosphate (NADP+) by a change in absorbance at
Figure 1. Schematic Diagram of Murine Sperm
(A) Sperm are highly polarized cells, divided into two main structural and functional units, the head and flagellum. The flagellum is further compartmentalized into
the midpiece, principal piece, and small terminal endpiece (not labeled). Oxidative phosphorylation is restricted to the midpiece where mitochondria are located.
The enzymes of glycolysis are located in the principal piece.
(B) Cross section of principal piece. Mammalian sperm have accessory structures known as the outer dense fibers (ODF) and the fibrous sheath (FS), which both
surround the central axoneme (9+2 microtubule doublets).
Chemistry & Biology
Sequential Enzyme Activities on a Hybrid Device
1014 Chemistry & Biology 16, 1013–1020, September 25, 2009 ª2009 Elsevier Ltd All rights reserved
340 nm. For both reactions, exogenous glucose-6-phosphate
dehydrogenase (G6PDH) was added (see Experimental Proce-
dures). We confirmed that the changes in absorbance specifi-
cally measured the target enzyme activities by running control
reactions in the absence of exogenous G6PDH or substrate,
and comparing the values from the experimental proteins versus
commercially available purified HK1 and GPI.
His-HK demonstrated activity without the germ-cell-targeting
region, with a Vmaxequal to 12 U/mg and a Kmequal to 0.27 mM
(glucose) (Table 1). These results were very similar to the values
for the commercial HK enzyme in solution (Vmax= 12 U/mg, Km=
0.25 mM glucose). These results demonstrated that our re-
combinant HK remained functional with the germ-cell-specific
domain replaced by the His-tag. We next evaluated the activity
of His-HK when adsorbed to a 1 cm2gold surface modified to
contain Ni-NTA groups (Figure 3A). The recombinant showed
activity on the surface, with negative controls being the recombi-
nant on the chip in the absence of the exogenous reaction
components, and also a blank Ni-NTA chip in the presence of
the reaction components. To identify the value of co-opting the
germ-cell-specific targeting domain, we made several attempts
to generate a recombinant HK lacking either the native targeting
domain or the His-tag (by means of the enterokinase site
provided by the vector; data not shown). These proteins were
highly labile and their degradation prevented us from obtaining
interpretable comparative data.
We utilized a similar approach with His-GPI, which had a Vmax
equal to 49 U/mg and a Kmequal to 0.23 mM (fructose 6-phos-
phate) when tested in solution (Table 1). Unlike the HK, we were
able to utilize enterokinase (Novagen, EMD/Merck, Madison, WI)
to remove the His-tag and still yield a stable protein. We used
enterokinase resin and Ni-NTA agarose to remove the residual
enterokinase and the protein from the cleaved His-tag, and veri-
immunoblotting using an antibody against the His-tag. After the
cleavage, wemeasured the kineticactivity of theGPI proteinand
found similar results as prior to cleavage. The GPI construct
without the His-tag had a Vmaxequal to 51 U/mg, and a Kmequal
to 0.31 mM (fructose 6-phosphate). As with the HK, these values
were very similar to those obtained for the commercial enzyme
(Vmax= 54 U/mg, Km= 0.48 mM fructose 6-phosphate). Both
experimental and control GPI recombinant proteins were stable
for more than 2 months when stored at 4?C.
We next evaluated the activity of the His-GPI (Figure 3B) when
tethered to Ni-NTA gold surfaces. The recombinant showed
activity when adsorbed to the surface, indicating that the protein
was functional even when tethered. The adsorption and specific
activities on the chips were significantly different between His-
GPI and the control GPI, which had the His-tag removed with
enterokinase (Figure 4). Thus, the amino-terminal attachment
not only facilitated a higher amount of total protein bound to
the surface, but also significantly improved the activity of the
protein that was adsorbed versus an equal amount adsorbed
His-HK and His-GPI Reactions in Series
on a Solid Surface
After verifying the activities of the individual enzymes, we next
mixed the recombinant His-HK and His-GPI in several different
molar ratios and attached them to the same Ni-NTA modified
surface. These two enzymes represent the first two sequential
steps of glycolysis. After immobilization and thorough rinsing,
Figure 2. Design of Recombinant Proteins
and Verification of Purified His-HK and
(A) Hexahistidine tags were introduced to the N
termini of HK and GPI. The known germ-cell-
specific targeting domain of HK1-sc was directly
replaced by the His-tag.
(B and C) SDS-PAGE and immunoblot analysis of
His-HK (B) and His-GPI (C) were performed as
a quality control of the purification process and
to verify the identity of the products. Coomassie
brilliant blue(CBB) staining wasperformed tovisu-
alize the total proteins that were present at the end
of purification. An antibody against the His-tag
(His) indicated the apparent molecular weights of
HK (HK) and GPI (GPI) were used to confirm the
identity of the enzymes.
Table 1. Properties of Recombinant Proteins
His-HK 12 ± 5.8 0.27 ± 0.12
His-GPI49 ± 8.9 0.23 ± 0.09
Enzyme activities in solution were determined as described. Values are
for the substrates glucose (mM) for HK, and fructose 6-phosphate
(mM) for GPI. The values shown are means and standard deviations
(three protein preparations for each).
Chemistry & Biology
Sequential Enzyme Activities on a Hybrid Device
Chemistry & Biology 16, 1013–1020, September 25, 2009 ª2009 Elsevier Ltd All rights reserved 1015
we tested for sequential enzymatic activities of the tethered re-
combinant proteins. We found that the two enzymes did in fact
act in series, metabolizing glucose into fructose 6-phosphate
(Figure 5). In our experiments, we found the His-HK protein to
be rate limiting (data not shown). Controls included adsorbing
His-HK alone with the complete reagents for the coupled reac-
tion pathway, and His-GPI alone with the complete reagents
for the coupled reaction pathway, and with the buffer alone. All
controls gave equivalent baseline readings (Figure 5), confirming
that the change in absorbance we observed did result from the
sequential activities of the tethered enzymes. Only the two
enzymes together showed activity under the coupled reaction
pathway we employed. This demonstrated that the reaction
Figure 3. Activities of Bound His-HK and
His-GPI When Tethered Individually
The recombinant proteins were tethered to gold
chips with Ni-NTA surfaces, and washed well to
remove any loosely bound or unbound proteins.
Coupled enzymatic reactions were used to deter-
mine the activities of the proteins bound to the
supports, with a change in absorbance at
340 nm measured spectrophotometrically, as de-
scribed. Activities represent means from nine
samples, with three samples from each of three
protein preparations. Bars indicate the standard
buffer and its individual components
were not contaminated with either of the
enzymes, nor other chemicals that could
cause a change in absorbance and
a false-positive read out of sequential
Tethering proteins to solid supports can
interfere with function in several ways.
The simple addition of a binding tag can
induce folding errors that reduce activity
(Halliwell et al., 2001), or can block sites
critical for substrate or cofactor binding,
or can prevent needed conformational
changes. In the case of HK, which is
well-characterized in terms of domain
structure and function, one can appre-
ciate the complexity of this problem.
The approximately 100 kDa isoforms of
thisprotein likely arosefroma duplication
and fusion event of an ancestral 50 kDa
form, and catalytic function is associated
with the C terminus, whereas regulatory
function is associated with the N ter-
placement of a fusion tag might interfere
with ATP, glucose, or glucose-6-phos-
phate binding sites, the locations of
which have been modeled based on data from crystals of the
rat brain HK1 (Mulichak et al., 1998).
The N-terminal germ-cell-specific sequence in HK1 that func-
tions as a targeting domain (Mori et al., 1993; Travis et al., 1999)
provides insight into how this protein could be tethered without
retarding its function. Removal of the His-tag from His-HK re-
sulted in an unstable protein, precluding comparisons of HK
activity with and without the His-tag. However, His-HK had
approximately the same activity in solution as the commercially
available enzyme, demonstrating that replacement of the target-
ing domain with the His-tag was not significantly detrimental to
its function. Copying the amino-terminal location of the germ-
cell-specific domain of HK proved effective for His-GPI as well.
Chemistry & Biology
Sequential Enzyme Activities on a Hybrid Device
1016 Chemistry & Biology 16, 1013–1020, September 25, 2009 ª2009 Elsevier Ltd All rights reserved
This recombinant protein showed almost a 9-fold improvement
in specific activity when immobilized via the His-tag versus
a control GPI without the His-tag that was randomly adsorbed
to the surface.
Although an N-terminal germ-cell-specific sequence has not
to date been identified for GPI, differences in electrophoretic
patterns for a germ cell variant have been reported (Buehr and
McLaren, 1981). Other germ-cell-specific glycolytic isozymes
have been described that differ either in sequence, activity, or
other characteristics for aldolase (Gillis and Tamblyn, 1984),
triose-phosphate isomerase (Auer et al., 2004), glyceraldehyde
3-phosphate dehydrogenase (GAPD-S) (Welch et al., 1992),
phosphoglycerate kinase (Boer et al., 1987), enolase (Edwards
and Grootegoed, 1983), and pyruvate kinase (Feiden et al.,
2007), as well as for the extra-glycolytic but related enzyme
lactate dehydrogenase (LDH-C4) (Zhong and Kleene, 1999)
Molecular characterization of these isozymes to identify
potential germ-cell-specific targeting domains might be of prac-
tical use in recapitulating the full glycolytic pathway on hybrid
organic-inorganic devices. Completion of a functional, immobi-
lized glycolytic pathway might facilitate the development of
internal medical applications for hybrid devices, because they
might one day be able to generate their own ATP locally from
circulating glucose. Until that time, the present success with
the first two enzymes of the pathway should provide a model
system for the study of various questions related to enzyme
activity on surfaces. For example, the resolution of lithographic
patterning has made rapid advancements, and having enzymes
might impact the efficiency of diffusion or substrate channelling.
To the best of our knowledge, these data represent the first
demonstration that sequential steps of a biological pathway
to a single, solid support. The pathway in question, glycol-
ysis, metabolizes glucose to pyruvate, releasing energy
that can be used to produce ATP. Thus, although these first
enzymes be reconstituted on a single device in the future,
such a system could utilize circulating glucose as a meta-
bolic substrate to generate ATP for a variety of functions.
This would be of practical importance for the development
of technical applications such as hybrid organic-inorganic
medical devices. Progress toward such goals is limited by
technical challenges; for example, tethering proteins to
a support can interfere with function in a number of ways.
This obstacle was overcome by mimicking the design of
sperm, in which specific isoforms of glycolytic enzymes
are bound to the fibrous sheath, and thus naturally attached
to a solid interface. Knowledge of the germ-cell-specific
targeting domain of HK1-sc allowed identification of a site
through which HK could be immobilized and still retain func-
tion. Recombinant His-HK and His-GPI were active individu-
ally and in series, with the activity of His-HK proving to be
rate limiting. His-GPI immobilized using this oriented
Figure 4. Use of the Amino-Terminal His-tag
Ni-NTA attachment chemistry improved binding and the specific activity of the GPI relative to GPI randomly adsorbed to the surface. The His-tag was cleaved
from His-GPI by means of an enterokinase site provided by the vector.
(A) Approximately twice as much His-GPI bound to a Ni-NTA support in comparison with control GPI lacking the His-tag (p = 0.012, paired Student’s t test; nine
samples, with three samples from each of three protein preparations. Bars indicate the standard deviations).
(B)Normalizing forthe amount of protein bound allowed aquantificationof thespecificactivities of theadsorbed proteins. Thespecific activityof theHis-GPI was
approximately nine times greater than that of the randomly absorbed GPI, although the enzyme activities in solution were relatively equal (p = 0.006, paired
Student’s t test; nine samples, with three samples from each of three protein preparations. Bars indicate the standard deviations).
Chemistry & Biology
Sequential Enzyme Activities on a Hybrid Device
Chemistry & Biology 16, 1013–1020, September 25, 2009 ª2009 Elsevier Ltd All rights reserved 1017
attachment chemistry had a significantly higher specific
activity than did GPI that was randomly adsorbed to the
surface, demonstrating the importance of oriented immobi-
lization. Additional work will be needed to generate and test
other pathway components, and also to determine the
optimal ratios and patterning of the enzymes to maximize
flux through the system and minimize the diffusion of meta-
Generation of Recombinant Hexokinase
and Glucose-6-Phosphate Isomerase
using the following primers, 50-ATGGGACAGAACTGCCAGCGAGGAC-30
(forward, HK1-sc), 50- TTAGGCGTTCGTAGGGTCTCCTCTGAGCC-30(re-
verse, HK1-sc), 50- ATGGCTGCGCTCACCCGGAACC-30(forward, GPI), and
50- TTATTCTAGTTTGGTGTCCCGCTGTTGC-30(reverse, GPI). The germ-cell-
specific targeting domain of HK1-sc was removed by nested PCR using
another forward primer, 50- GAAAAGATTGATAAGTATCTGTATGCCATGCG
Max TOPO TA (Invitrogen, Carlsbad, CA), which added a 6 histidine repeat on
the amino terminus, followed by an enterokinase cleavage site and then the
glycolytic enzyme’s sequence. Constructs were validated by sequencing and
then were transfected into HEK293-F-FreeStyle cells (Invitrogen), using the
293fectin transfection reagent (Invitrogen). The protein was purified from cell
lysates 48 hr later using a Ni-NTA purification system (Invitrogen). The purified
proteins were dialyzed three times with 1 l 50 mM MOPS, and then concen-
trated using Amicon filtration centritubes (Millipore, Billerica, MA). Protein
concentrations were determined with the Micro-BCA assay (Pierce, Rockford,
The primary antibodies used were as follows: mouse anti-His tag (1:5000
dilution, Invitrogen), mouse anti-type1 hexokinase (1:1000 dilution, Chemicon,
Temecula, CA), and rabbit anti-GPI (1:500 dilution, Santa Cruz Biotechnology,
GE Healthcare, Piscataway, NJ).
Preparation of Ni-NTA Surface
16-mercaptohexadecanoic acid (MHA), ethanol, trifluoroacetic anhydride
(TFA), triethylamine (TEA), N, N-dimethylformamide (DMF), tetrahydrofuran
(THF), sodium hydroxide (NaOH), ethylenediaminetetraacetic acid (EDTA),
nickel chloride (NiCl2), N, N-bis-(carboxymethyl)-L-lysine-hydrate (NTA),
triethylene glycol, and all other reagents were purchased from Sigma-Aldrich
(St. Louis, MO) unless otherwise indicated. Gold surfaces (10/100 nm Cr/Au
deposited on silicon by electron-beam evaporation) were made functional
with Ni-NTA for the immobilization of His-tagged proteins as described previ-
ously (Yan et al., 1997) with some modifications. Briefly, gold surfaces were
cleaned by incubating in a mixture of 2:1 H2SO4:H2O2(a highly corrosive/
oxidizing solution) for 5 min, followed by rinsing with deionized water and
then ethanol. Samples were immediately immersed in an ethanol solution con-
taining 2 mM MHA and incubated overnight (?16 hr). Control surfaces were
incubated in ethanol without MHA. After incubation the surfaces were rinsed
with ethanol, blown dry with high purity nitrogen (99.999%), and subsequently
immersed in a solution of 100 mM TFA and 200 mM TEA in anhydrous DMF for
30 min. After the reaction (in which interchain anhydrides were formed),
and 7.5 mM NaOH at room temperature for 30 min. The surfaces were then
rinsed with deionized water and blown dry under a stream of nitrogen. Ni-
NTA activation was performed by immersing the gold surface in a solution of
40 mM NiCl2at room temperature for 2 hr. Finally, the functionalized gold
surface was rinsed thoroughly with deionized water, dried with nitrogen, and
cut into pieces (?1 cm2) using a diamond-tipped stylus.
Spectrophotometric Analysis of His-HK and His-GPI Activity
HK and GPI activities were measured by means of coupled enzyme reaction
pathways that led to the reduction of NADP+to NADPH. The rate of this reduc-
tion was measured as a change in absorbance at 340 nm using a spectropho-
tometer (SAFIRE microplate reader, Tecan, Medford, MA). The coupled
reactions for HK were as follows: glucose + ATP / glucose-6-phosphate +
ADP and glucose-6-phosphate + NADP+/ NADPH + 6-phospho-glucono-
d-lactone, with the first reaction catalyzed by His-HK, and the second cata-
lyzed by exogenous G6PDH. The coupled reactions for GPI were as follows:
fructose-6-phosphate / glucose-6-phosphate and glucose-6-phosphate +
NADP+/ NADPH + 6-phospho-glucono-d-lactone, with the first reaction
catalyzed by His-GPI, and the second catalyzed by exogenous G6PDH. Fresh
stocks of NADP+and ATP were prepared immediately prior to each trial. The
base reaction mixture contained 1 mM NADP+, 10 mM MgCl2, and 3 U/ml
G6PDH in 50 mM MOPS buffer (pH 7.4). To quantify HK activity, 1 mM ATP
and 5 mM glucose were added. To quantify GPI activity, 5 mM fructose
6-phosphate was added. Measurements of activity were determined from
Figure 5. Activity of His-HK and His-GPI in Series on a Single
(A) Schematic diagram of surface chemistry, recombinant proteins, and the
enzymatic reactions. The enzymes were immobilized to a solid support via
the interaction of the His-tags with Ni-NTA.
(B) His-HK and His-GPI were tethered together, allowing glucose to be cata-
lyzed sequentially to glucose-6-phosphate and then to fructose-6-phosphate.
Completion of the reaction was detected by the coupled reaction pathway as
described. ‘‘HK-GPI’’ denotes supports incubated with 0.5 nM His-HK and
0.5 nM His-GPI. Readings for supports incubated with His-HK alone (HK) or
His-GPI alone (GPI) were taken with protein solutions at 1 nM concentration.
A blank was performed with the Ni-NTA surface incubated in MOPS buffer
(buffer). This figure is a representative result from one set of protein prepara-
tions, with triplicate samples (bars denote standard deviations). Because
binding might be predicted to differ slightly for each protein for each set of
preparations, it would not be valid to pool the data for the experiments;
however, the experiment was performed with three separate sets of prepara-
tions with very similar results each time.
Chemistry & Biology
Sequential Enzyme Activities on a Hybrid Device
1018 Chemistry & Biology 16, 1013–1020, September 25, 2009 ª2009 Elsevier Ltd All rights reserved
slopes taken from within the linear range. The Kmand Vmaxfor each enzyme
were calculated by quantifying the enzymatic activities at various concentra-
tions of their respective substrates and fitting the data to hyperbolic functions.
One unit of enzyme activity was defined as the amount of enzyme that cata-
lyzed the formation of 1 mmol NADPH per minute.
Tethering of Enzymes to Inorganic Supports and Quantification
of Activity while Bound
To determine the amount of protein immobilized to the gold surfaces, we
measured the protein concentration before and after surface adsorption using
the Micro-BCA assay according to the manufacturer’s instructions. The
proteins were adsorbed by placing 60 ml 50 mM MOPS (pH 7.4) containing
known amounts of protein, at room temperature in the dark on a single gold
Ni-NTA surface (1 3 1cm square). After 15 min, 50 ml solution was removed
by pipetting, and 90 ml MOPS buffer was gently added to the surface. This
volume was then collected and combined with the 50 ml that had been
removed initially. The 140 ml was then analyzed to measure the amount of
protein in solution that did not bind to the surface. Two more washes with
90 ml volumes of the MOPS buffer were performed, and these were also
collected individually for quantification of protein that was removed. The
amount of immobilized protein was calculated by subtracting the amount of
protein found in the rinsing solutions from the initial starting amount. It should
be noted that the amounts of protein removed by the second and third washes
were consistently below the levels of detection. To ensure that all unbound or
loosely bound protein wasremoved,the surface wasthengently washed three
times with 5 ml MOPS buffer. Thus, it should be noted that for the purposes of
quantification of specific activity, the amounts of protein actually bound were
equal to or less than the amounts quantified, giving a conservative error in
terms of specific activity.
and after adsorption of the HK/GPI protein solution using imaging ellipsometry
(SE-EP3, Nanofilm, Goettingen, Germany). At least three different locations on
a surface were measure and analyzed. Film thickness values were calculated
using a two-layer optical model (layer 1 = substrate and layer 2 = organic film)
using the Thinfilm Companion software (Semiconsoft, Boston, MA), where
experimental data were fitted using a modified Marquardt-Levenberg minimi-
zation. The optical properties of the gold substrate (layer 1) were calculated
using cleaned gold surfaces. Self-assembled monolayers and protein layers
(layer 2) were modeled using a refractive index (n, k) of n = 1.45 and k = 0.
The optical thickness of HK/GPI (with or without the His-tag) adsorbed to
a Ni-NTA surface is reported in Figure S1 (available online). Adsorbed His-
tagged protein showed a thickness increase of ?3.2 nm compared with the
Ni-NTA surface whereas the protein without the tag only measured a layer
thickness of ?0.65 nm. We also rinsed the surfaces briefly with a 10 mM
EDTA solution (pH 8) to evaluate desorption of immobilized His-tagged
protein. The EDTA rinse resulted in a ?50% reduction of His-tagged protein
layer thickness whereas non-His-tagged protein showed similar results as
before rinsing. These results indicate that the proteins most likely were
oriented on the surface via their His-tag and that the binding to some extent
The enzyme reactions themselves were carried out in a well with the chip
in a 1 ml volume of reaction mixture. The changes in absorbance at 340 nm
at various time points were measured in 300 ml aliquots of reaction mixture
that were removed and transferred to another well within the plate. Readings
were obtained using a spectrophotometer as above. One unit of enzyme
activity was defined as the amount of enzyme that catalyzed the formation
of 1 mmol NADPH per min. All assays were performed at least three times,
each with at least triplicate sets of chips. Negative controls included the
Ni-NTA surface alone and the surface with adsorbed protein, but missing
the exogenous substrate. When possible, comparative measurements were
obtained using chips adsorbed with the His-tagged recombinants versus
recombinants that had the His-tag removed by enterokinase cleavage.
To determine whether His-HK and His-GPI could act in series, the proteins
were immobilized by placing 60 ml of mixed protein solution (0.5 nM His-HK,
0.5 nM His-GPI in 50 mM MOPS buffer [pH 7.4]) at room temperature in the
dark on a single gold Ni-NTA surface (1 3 1cm square). After 15 min, 50 ml
protein solution was removed by pipetting, and a similar washing procedure
and protein assay were performed as described above. The assay of the
sequential reaction of HK-GPI was performed at room temperature by
measuring the oxidation of NADH at 340 nm as a result of exogenous glycerol
3-phosphate dehydrogenase. The reaction was carried out in a similar fashion
as those for HK and GPI individually, with the reaction mixture containing
50 mM MOPS, 5 mM glucose, 1 mM ATP, 0.5 mM NADH, 2 mM MgCl2,
0.5 U/ml phosphofructokinase, 0.5 U/ml aldolase, 7 U/ml triose phosphate
isomerase, and 2 U/ml glycerol 3-phosphate dehydrogenase at pH 7.4. Fresh
stocks of NADH and ATP were prepared immediately prior to each trial. One
unit of coupled reaction activity was defined as the amount of enzyme that
catalyzed the formation of 1 mmol NAD+per min. All assays were performed
atleastthree times,eachwithatleasttriplicate setsofchips. Negative controls
included the Ni-NTA surface alone, the surface with both adsorbed proteins
but missing the exogenous substrate, or His-HK and the pathway reagents,
or His-GPI and the pathway reagents.
Supplemental Data include a figure and can be found with this article
online at http://www.cell.com/chemistry-biology/supplemental/S1074-5521
We thank Anita Hesser and Jane Miller for their outstanding administrative
support. This work was primarily supported by an Innovation Award from the
Cornell Institute for Biotechnology and Life Science Technologies, a NYSTAR
Designated Center for Advanced Technology (A.J.T.); other support was
provided by the Marilyn M. Simpson Faculty Career Development Award
(A.J.T.), the National Institutes of Health R01-HD045664 (A.J.T.), and the
faculty research awards program (FRAP-A) at the University of Albany
(M.B.). Part of this work was performed using facilities and equipment sup-
ported by the STC Program of the National Science Foundation under Agree-
ment No. ECS-9876771.
Received: February 16, 2009
Revised: July 24, 2009
Accepted: August 5, 2009
Published: September 24, 2009
Auer, J., Camoin, L., Courtot, A.M., Hotellier, F., and De Almeida, M. (2004).
Evidence that P36, a human sperm acrosomal antigen involved in the fertil-
ization process is triosephosphate isomerase. Mol. Reprod. Dev. 68, 515–
Basso, A., Braiuca, P., Ebert, C., Gardossi, L., and Linda, P. (2006). Properties
and applications of supports for enzyme-mediated transformations in solid
phase synthesis. J. Chem. Technol. Biotechnol. 81, 1626–1640.
Boer, P.H., Adra, C.N., Lau, Y.F., and McBurney, M.W. (1987). The testis-
specific phosphoglycerate kinase gene pgk-2 is a recruited retroposon. Mol.
Cell. Biol. 7, 3107–3112.
tion of glucosephosphate isomerase in mouse spermatozoa. J. Reprod. Fertil.
Cao, W., Gerton,G.L., and Moss, S.B. (2006). Proteomic profiling of accessory
structures from the mouse sperm flagellum. Mol. Cell. Proteomics 5, 801–810.
Du, Y.Z., Hiratsuka, Y., Taira, S., Eguchi, M., Uyeda, T.Q., Yumoto, N., and
ATP. Chem. Commun. (Camb.) 16, 2080–2082.
Edwards, Y.H., and Grootegoed, J.A. (1983). A sperm-specific enolase.
J. Reprod. Fertil. 68, 305–310.
Feiden, S., Stypa, H., Wolfrum, U., Wegener, G., and Kamp, G. (2007). A novel
pyruvate kinase (PK-S) from boar spermatozoa is localized at the fibrous
sheath and the acrosome. Reproduction 134, 81–95.
Chemistry & Biology
Sequential Enzyme Activities on a Hybrid Device
Chemistry & Biology 16, 1013–1020, September 25, 2009 ª2009 Elsevier Ltd All rights reserved 1019
Fischer, T., and Hess, H. (2007). Materials chemistry challenges in the design
of hybrid bionanodevices: supporting protein function within artificial environ-
ments. J. Mater. Chem. 17, 943–951.
Gillis, B.A., and Tamblyn, T.M. (1984). Association of bovine sperm aldolase
with sperm subcellular components. Biol. Reprod. 31, 25–35.
Halliwell, C.M., Morgan, G., Ou, C.P., and Cass, A.E. (2001). Introduction of
a (poly)histidine tag in L-lactate dehydrogenase produces a mixture of active
and inactive molecules. Anal. Biochem. 295, 257–261.
Krisfalusi, M., Miki, K., Magyar, P.L., and O’Brien, D.A. (2006). Multiple glyco-
lytic enzymes are tightly bound to the fibrous sheath of mouse spermatozoa.
Biol. Reprod. 75, 270–278.
Laurent, N., Haddoub, R., and Flitsch, S.L. (2008). Enzyme catalysis on solid
surfaces. Trends Biotechnol. 6, 328–337.
Luo, T.J., Soong, R., Lan, E., Dunn, B., and Montemagno, C. (2005). Photo-
induced proton gradients and ATP biosynthesis produced by vesicles encap-
sulated in a silica matrix. Nat. Mater. 4, 220–224.
Miki, K., Qu, W., Goulding, E.H., Willis, W.D., Bunch, D.O., Strader, L.F.,
Perreault, S.D., Eddy, E.M., and O’Brien, D.A. (2004). Glyceraldehyde 3-phos-
phate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for
sperm motility and male fertility. Proc.Natl. Acad.Sci.USA 101,16501–16506.
Mori, C., Welch, J.E., Fulcher, K.D., O’Brien, D.A., and Eddy, E.M. (1993).
Unique hexokinase messenger ribonucleic acids lacking the porin-binding
domain are developmentally expressed in mouse spermatogenic cells. Biol.
Reprod. 49, 191–203.
Mori, C., Nakamura, N., Welch, J.E., Gotoh, H., Goulding, E.H., Fujioka, M.,
and Eddy, E.M. (1998). Mouse spermatogenic cell-specific type 1 hexokinase
(mHk1-s)transcriptsareexpressed byalternative splicingfromthemHk1 gene
and the HK1-S protein is localized mainly in the sperm tail. Mol. Reprod. Dev.
Mukai, C., and Okuno, M. (2004). Glycolysis plays a major role for adenosine
triphosphate supplementation in mouse sperm flagellar movement. Biol.
Reprod. 71, 540–547.
Mulichak, A.M., Wilson, J.E., Padmanabhan, K., and Garavito, R.M. (1998).
The structure of mammalian hexokinase-1. Nat. Struct. Biol. 5, 555–560.
Schoemaker, H.E., Mink, D., and Wubbolts, M.G. (2003). Dispelling themyths–
biocatalysis in industrial synthesis. Science 299, 1694–1697.
Steinberg-Yfrach, G., Rigaud, J.L., Durantini, E.N., Moore, A.L., Gust, D., and
Moore, T.A. (1998). Light-driven production of ATP catalysed by F0F1-ATP
synthase in an artificial photosynthetic membrane. Nature 392, 479–482.
Storey, B.T., and Kayne, F.J. (1975). Energy metabolism of spermatozoa. V.
The Embden-Myerhof pathway of glycolysis: activities of pathway enzymes
in hypotonically treated rabbit epididymal spermatozoa. Fertil. Steril. 26,
Tachibana, S.,Suzuki, M.,and Asano,Y. (2006). Application ofan enzymechip
to the microquantification of l-phenylalanine. Anal. Biochem. 359, 72–78.
Travis, A.J., and Kopf, G.S. (2002). The spermatozoon as a machine: compart-
mentalized pathwaysbridgecellular structure and function. In Assisted Repro-
ductive Technology: Accomplishments and New Horizons, C.J. De Jonge and
C.L. Barratt, eds. (New York: Cambridge University Press), pp. 26–39.
Travis, A.J., Foster, J.A., Rosenbaum, N.A., Visconti, P.E., Gerton, G.L., Kopf,
G.S., and Moss, S.B. (1998). Targeting of a germ cell-specific type 1 hexoki-
nase lacking a porin- binding domain to the mitochondria as well as to the
head and fibrous sheath of murine spermatozoa. Mol. Biol. Cell 9, 263–276.
Travis, A.J., Jorgez, C.J., Merdiushev, T., Jones, B.H., Dess, D.M., Diaz-
Cueto, L., Storey, B.T., Kopf, G.S., and Moss, S.B. (2001). Functional relation-
ships between capacitation-dependent cell signaling and compartmentalized
metabolic pathways in murine spermatozoa. J. Biol. Chem. 276, 7630–7636.
Travis, A.J., Sui, D., Riedel, K.D., Hofmann, N.R., Moss, S.B., Wilson, J.E., and
Kopf, G.S. (1999). A novel NH(2)-terminal, nonhydrophobic motif targets
a male germ cell-specific hexokinase to the endoplasmic reticulum and
plasma membrane. J. Biol. Chem. 274, 34467–34475.
Ummat, A., Dubey, A., and Marvoidis, C. (2005). Biol.-Nanorobotics: A field
inspired by Nature. In Biomimetics: Biologically Inspired Technologies, Y.
Bar-Cohen, ed. (Boca Raton, FL, USA: CRC Press), pp. 201–227.
Welch, J.E., Schatte, E.C., O’Brien, D.A., and Eddy, E.M. (1992). Expression of
a glyceraldehyde 3-phosphate dehydrogenase gene specific to mouse sper-
matogenic cells. Biol. Reprod. 46, 869–878.
Westhoff, D., and Kamp, G. (1997). Glyceraldehyde 3-phosphate dehydroge-
nase is bound to the fibrous sheath of mammalian spermatozoa. J. Cell Sci.
Wilson, J.E. (1997). An introduction to the isoenzymes of mammalian hexoki-
nase types I-III. Biochem. Soc. Trans. 25, 103–107.
Yan, L., Marzolin, C., Terfort, A., and Whitesides, G. (1997). Formation and
reaction of interchain carboxylic anhydride groups on self-assembled mono-
layers on gold. Langmuir 13, 6704–6712.
Zhong, X., and Kleene, K.C. (1999). cDNA copies of the testis-specific lactate
processed pseudogenes are not derived from mRNAs that are expressed in
haploid and late meiotic spermatogenic cells. Mamm. Genome 10, 6–12.
Zinkham, W.H. (1968). Lactate dehydrogenase isozymes of testis and sperm:
biological and biochemical properties and genetic control. Ann. N Y Acad. Sci.
Chemistry & Biology
Sequential Enzyme Activities on a Hybrid Device
1020 Chemistry & Biology 16, 1013–1020, September 25, 2009 ª2009 Elsevier Ltd All rights reserved