K Brocklehurst

Queen Mary, University of London, London, ENG, United Kingdom

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Publications (179)644.67 Total impact

  • Keith Brocklehurst, Mike P Philpott
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    ABSTRACT: Desquamation or cell shedding in mammalian skin is known to involve serine proteases, aspartic proteases and glycosidases. In addition, evidence continues to accumulate that papain-like cysteine proteases and an inhibitor cystatin M/E largely confined to the cutaneous epithelia also play key roles in the process. This involves the complete proteolysis of cell adhesive structures of the stratum corneum, the corneodesmosomes and notably of the desmogleins. Continual cell replacement in the epidermis is the result of the balance between the loss of the outer squames and mitosis of the cells in the basal cell layer. This article provides a brief account of the salient features of the characteristics and catalytic mechanism of cysteine proteases, followed by a discussion of the relevant epidermal biology. The proteases include the asparaginyl endopeptidase legumain, which exerts a strict specificity for the hydrolysis of asparaginyl bonds, cathepsin-V and cathepsin-L. The control of these enzymes by cystatin M/E regulates the processing of transglutaminases and is crucial in the biochemical pathway responsible for regulating the cross-linking and desquamation of the stratum corneum. In addition, caspase-14 has now been shown to play a major part in epidermal maturation. Uncontrolled proteolytic activity leads to abnormal hair follicle formation and deleterious effects on the skin barrier function.
    Cell and Tissue Research 01/2013; · 3.68 Impact Factor
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    ABSTRACT: Understanding the roles of noncovalent interactions within the enzyme molecule and between enzyme and substrate or inhibitor is an essential goal of the investigation of active center chemistry and catalytic mechanism. Studies on members of the papain family of cysteine proteinases, particularly papain (EC 3.4.22.2) itself, continue to contribute to this goal. The historic role of the catalytic site Cys/His ion pair now needs to be understood within the context of multiple dynamic phenomena. Movement of Trp177 may be necessary to expose His159 to solvent with consequent decrease in its degree of electrostatic solvation of (Cys25)-S(-). Here we report an investigation of this possibility using computer modeling of quasi-transition states and pH-dependent kinetics using 3,3'-dipyridazinyl disulfide, its n-propyl and phenyl derivatives, and 4,4'-dipyrimidyl disulfide as reactivity probes that differ in the location of potential hydrogen-bonding acceptor atoms. Those interactions that influence ion pair geometry and thereby catalytic competence, including by transmission of the modulatory effect of a remote ionization with pK(a) 4, were identified. A key result is the correlation between the kinetic influence of the modulatory trigger of pK(a) 4 and disruption of the hydrogen bond donated by the indole N-H of Trp177, the hydrophobic shield of the initial "intimate" ion pair. This hydrogen bond is accepted by the amide O of Gln19-a component of the oxyanion hole that binds the tetrahedral species formed from the substrate during the catalytic act. The disruption would be expected to contribute to the mobility of Trp177 and possibly to the effectiveness of the binding of the developing oxyanion.
    Biochemistry 11/2011; 50(49):10732-42. · 3.38 Impact Factor
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    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    ChemInform 01/2010; 33(19).
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    ABSTRACT: Pectate lyases harness anti beta-elimination chemistry to cleave the alpha-1,4 linkage in the homogalacturonan region of plant cell wall pectin. We have studied the binding of five pectic oligosaccharides to Bacillus subtilis pectate lyase in crystals of the inactive enzyme in which the catalytic base is substituted with alanine (R279A). We discover that the three central subsites (-1, +1, and +2) have a profound preference for galacturonate but that the distal subsites can accommodate methylated galacturonate. It is reasonable to assume therefore that pectate lyase can cleave pectin with three consecutive galacturonate residues. The enzyme in the absence of substrate binds a single calcium ion, and we show that two additional calcium ions bind between enzyme and substrate carboxylates occupying the +1 subsite in the Michaelis complex. The substrate binds less intimately to the enzyme in a complex made with a catalytic base in place but in the absence of the calcium ions and an adjacent lysine. In this complex, the catalytic base is correctly positioned to abstract the C5 proton, but there are no calcium ions binding the carboxylate at the +1 subsite. It is clear, therefore, that the catalytic calcium ions and adjacent lysine promote catalysis by acidifying the alpha-proton, facilitating its abstraction by the base. There is also clear evidence that binding distorts the relaxed 2(1) or 3(1) helical conformation of the oligosaccharides in the region of the scissile bond.
    Biochemistry 12/2009; 49(3):539-46. · 3.38 Impact Factor
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    Keith Brocklehurst, Sheraz Gul, Richard W. Pickersgill
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    ABSTRACT: Closing a gap in the literature, this is the only series to cover this important topic in organic and biochemistry. Drawing upon the combined expertise of the international "who's who" in amino acid research, these volumes represent a real benchmark for amino acid chemistry, providing a comprehensive discussion of the occurrence, uses and applications of amino acids and, by extension, their polymeric forms, peptides and proteins.
    Amino acids, peptides and proteins in organic chemistry., Edited by A.B. Hughes, 10/2009: chapter Substrate Recognition.: pages 473-504; Wiley-VCH publishers.., ISBN: 3527320989
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    ABSTRACT: The extracellular proteinase produced by Porphyromonas gingivalis, which has often been described as ‘trypsin-like’, was isolated by thiol-disulphide interchange covalent chromatography. Evidence from the method of isolation, thiol-specific inhibition reactivation cycle and thiol-specific reactivity probe kinetics, all involving 2-pyridyl disulphides, compels the view that this enzyme, for which the name gingivain is here proposed, is a cysteine proteinase. All of the hydrolytic activity towards α-N-benzoyl-L-arginine-4-nitroanilide (L-BAPNA) and azocasein in the P. gingivalis vesicle/supernatant mixture was shown to be thiol dependent by its complete inhibition by 2,2′-dipyridyl disulphide and complete reactivation by 2-mercaptoethanol. The vesicles and all of the hydrolytic activity were isolated by precipitation in 70 per cent saturated ammonium sulphate, centrifugation for 22 h at 150,000 g and 4°C. The cysteine proteinase was prepared in fully active form by sequential elution covalent chromatography on Sepharose-glutathione 2-pyridyl disulphide gel, which separated the enzyme from the vesicles and from other thiol-containing protein devoid of catalytic activity towards L-BAPNA. The fully active isolated enzyme was: (a) completely inhibited by reaction with 2,2′-dipyridyl disulphide with complete reactivation by 2-mercaptoethanol, and (b) shown to possess a key catalytic site characteristic, typical of many cysteine proteinases. Thus, stopped-flow kinetic analysis of the reaction of its catalytically essential thiol group with 2,2′-dipyridyl disulphide showed the reactivity to be minimum at ca. pH 6, behaviour characteristic of the existence of a catalytic site cysteine-histidine interactive system.
    Microbial Ecology in Health and Disease 07/2009; 4(5):319-328.
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    ABSTRACT: Studies on papain (EC 3.4.22.2), the most thoroughly investigated member of the cysteine proteinase superfamily, have contributed substantially to our understanding of the roles of noncovalent interactions in enzyme active center chemistry. Previously, we reported evidence that the long-held view that catalytic competence develops synchronously with formation of the catalytic site (Cys25)-S-/(His159)-Im+H ion pair is incorrect and that conformational rearrangement is necessary for each of the partners to play its role in catalysis. A decrease in the level of mutual solvation of the partners of the noncatalytic "intimate" ion pair should release the nucleophilic character of (Cys25)-S- and allow association of (His159)-Im+H with the leaving group of a substrate to provide its general acid-catalyzed elimination. Hypotheses by which this could be achieved involve electrostatic modulation of the ion pair and perturbation of its hydrophobic shielding from solvent by Trp177. The potential electrostatic modulator closest to the catalytic site is Asp158, the mutation of which to Ala substantially decreases catalytic activity. Here we report an investigation of these hypotheses by a combination of computer modeling and stopped-flow pH-dependent kinetic studies using a new series of cationic aminoalkyl 2-pyridyl disulfide time-dependent inhibitors as reactivity probes. These probes 2-4 (n = 2-4), which exist as equilibrium mixtures of H3N+-[CH2]n-S-S-2-pyridyl+H and H3N+-[CH2]n-S-S-2-pyridyl which predominate in acidic and weakly alkaline media, respectively, were shown by modeling and kinetic analysis to bind with various degrees of effectiveness near Asp158 and in some cases also near Trp177. Kinetic analysis of the reactions of 2-4 and of the reaction of CH3-[CH2]2-S-S-2-pyridyl+H <==>CH3-[CH2]2-S-S-2-pyridyl 1 and normal mode calculations lead to the conclusion that Asp158 is not involved in the generation of nucleophilic character in the ion pair and demonstrates a key role for Trp177.
    Biochemistry 03/2008; 47(7):2025-35. · 3.38 Impact Factor
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    ABSTRACT: We provide a mechanism for the activity of pectin methylesterase (PME), the enzyme that catalyses the essential first step in bacterial invasion of plant tissues. The complexes formed in the crystal using specifically methylated pectins, together with kinetic measurements of directed mutants, provide clear insights at atomic resolution into the specificity and the processive action of the Erwinia chrysanthemi enzyme. Product complexes provide additional snapshots along the reaction coordinate. We previously revealed that PME is a novel aspartic-esterase possessing parallel beta-helix architecture and now show that the two conserved aspartates are the nucleophile and general acid-base in the mechanism, respectively. Other conserved residues at the catalytic centre are shown to be essential for substrate binding or transition state stabilisation. The preferential binding of methylated sugar residues upstream of the catalytic site, and demethylated residues downstream, drives the enzyme along the pectin molecule and accounts for the sequential pattern of demethylation produced by both bacterial and plant PMEs.
    The EMBO Journal 10/2007; 26(17):3879-87. · 9.82 Impact Factor
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    ABSTRACT: Ring contraction during cobalamin (vitamin B12) biosynthesis requires a seemingly futile methylation of the C20 position of the tetrapyrrole framework. Along the anaerobic route, this reaction is catalyzed by CbiL, which transfers a methyl group from S-adenosyl-L-methionine to cobalt factor II to generate cobalt factor III. CbiL belongs to the class III methyltransferases and displays similarity to other cobalamin biosynthetic methyltransferases that are responsible for the regiospecific methylation of a number of positions on the tetrapyrrole molecular canvas. In an attempt to understand how CbiL selectively methylates the C20 position, a detailed structure function analysis of the enzyme has been undertaken. In this paper, we demonstrate that the enzyme methylates the C20 position, that its preferred substrate is cobalt factor II, and that the metal ion does not undergo any oxidation change during the course of the reaction. The enzyme was crystallized, and its structure was determined by x-ray crystallography, revealing that the 26-kDa protein has a similar overall topology to other class III enzymes. This helped in the identification of some key amino acid residues (Asp(104), Lys(176), and Tyr(220)). Analysis of mutant variants of these groups has allowed us to suggest potential roles that these side chains may play in substrate binding and catalysis. EPR analysis of binary and ternary complexes indicate that the protein donates a fifth ligand to the cobalt ion via a gated mechanism to prevent transfer of the methyl group to water. The chemical logic underpinning the methylation is discussed.
    Journal of Biological Chemistry 09/2007; 282(33):23957-69. · 4.65 Impact Factor
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    ABSTRACT: A kinetically homogeneous anti-phosphate catalytic antibody preparation was shown to catalyse the hydrolysis of a series of O-aryl N-methyl carbamates containing various substituents in the 4-position of the O-phenyl group. The specific nature of the antibody catalysis was demonstrated by the adherence of these reactions to the Michaelis-Menten equation, the complete inhibition by a hapten analogue, and the failure of the antibody to catalyse the hydrolysis of the 2-nitrophenyl analogue of the 4-nitrophenylcarbamate substrate. Hammett sigma-rho analysis suggests that both the non-catalysed and antibody-catalysed reactions proceed by mechanisms in which development of the aryloxyanion of the leaving group is well advanced in the transition state of the rate-determining step. This is probably the ElcB (elimination-addition) mechanism for the non-catalysed reaction, but for the antibody-catalysed reaction might be either ElcB or B(Ac)2 (addition-elimination), in which the elimination of the aryloxy group from the tetrahedral intermediate has become rate-determining. This result provides evidence of the dominance of recognition of phenolate ion character in the phosphate hapten in the elicitation process, and is discussed in connection with data from the literature that suggest a B(Ac)2 mechanism, with rate-determining formation of the tetrahedral intermediate for the hydrolysis of carbamate substrates catalysed by an antibody elicited by a phosphonamidate hapten in which phenolate anion character is minimized. The present paper contributes to the growing awareness that small differences in the structure of haptens can produce large differences in catalytic characteristics.
    Biochemical Journal 03/2007; 401(3):721-6. · 4.65 Impact Factor
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    ABSTRACT: The temperature-dependences of the second-order rate constants (k) of the reactions of the catalytic site thiol groups of two cysteine peptidases papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) with a series of seven 2-pyridyl disulphide reactivity probes (R-S-S-2-Py, in which R provides variation in recognition features) were determined at pH 6.7 at temperatures in the range 4-30 degrees C by stopped-flow methodology and were used to calculate values of DeltaS++, DeltaH++ and DeltaG++. The marked changes in DeltaS++ from negative to positive in the papain reactions consequent on provision of increase in the opportunities for key non-covalent recognition interactions may implicate microsite desolvation in binding site-catalytic site signalling to provide a catalytically relevant transition state. The substantially different behaviour of actinidin including apparent masking of changes in DeltaH++ by an endothermic conformational change suggests a difference in mechanism involving kinetically significant conformational change.
    Biochemical Journal 06/2006; 396(1):17-21. · 4.65 Impact Factor
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    ABSTRACT: RALDH3 (retinal dehydrogenase 3) was characterized by kinetic and binding studies, protein engineering, homology modelling, ligand docking and electrostatic-potential calculations. The major recognition determinant of an RALDH3 substrate was shown to be an eight-carbon chain bonded to the aldehyde group whose kinetic influence (kcat/Km at pH 8.5) decreases when shortened or lengthened. Surprisingly, the b-ionone ring of all-trans-retinal is not a major recognition site. The dissociation constants (Kd) of the complexes of RALDH3 with octanal, NAD+ and NADH were determined by intrinsic tryptophan fluorescence. The similarity of the Kd values for the complexes with NAD+ and with octanal suggests a random kinetic mechanism for RALDH3, in contrast with the ordered sequential mechanism often associated with aldehyde dehydrogenase enzymes. Inhibition of RALDH3 by tri-iodothyronine binding in competition with NAD+, predicted by the modelling, was established kinetically and by immunoprecipitation. Mechanistic implications of the kinetically influential ionizations with macroscopic pKa values of 5.0 and 7.5 revealed by the pH-dependence of kcat are discussed. Analogies with data for non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans, together with the present modelled structure of the thioacyl RALDH3, suggest (a) that kcat characterizes deacylation of this intermediate for specific substrates and (b) the assignment of the pKa of the major ionization (approximating to 7.5) to the perturbed carboxy group of Glu280 whose conjugate base is envisaged as supplying general base catalysis to attack of a water molecule. The macroscopic pKa of the minor ionization (5.0) is considered to approximate to that of the carboxy group of Glu488.
    Biochemical Journal 03/2006; 394(Pt 1):67-75. · 4.65 Impact Factor
  • CSH protocols. 01/2006; 2006(1).
  • CSH protocols. 01/2006; 2006(1).
  • CSH protocols. 01/2006; 2006(1).
  • CSH protocols. 01/2006; 2006(1).
  • CSH protocols. 01/2006; 2006(1).
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    ABSTRACT: The substrate selectivities of an anti-phosphonate and an anti-phosphate kinetically homogeneous polyclonal catalytic antibody preparation and two hydrolytic enzymes were compared by using hapten-analogous and truncated carbonate and ester substrates each containing a 4-nitrophenolate leaving group. Syntheses of the truncated substrates devoid of recognition features in the non-leaving group parts of the substrates are reported. The relatively high kinetic selectivity of the more active anti-phosphonate antibody preparation is considered to depend on a relatively rigid catalytic site with substantial reaction centre specificity together with other important recognition interactions with the extended non-leaving group part of the substrate. In contrast, the less catalytically active, more flexible anti-phosphate antibody exhibits much lower kinetic selectivity for the substrate reaction centre comparable with that of the hydrolytic enzymes with activity much less dependent on recognition interactions with the non-leaving group part of the substrate. The ways in which haptenic flexibility and IgG architecture might contribute to the differential kinetic selectivities are indicated.
    Biochemical Journal 08/2004; 381(Pt 1):125-30. · 4.65 Impact Factor
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    ABSTRACT: The effects of increasing the content of the aprotic dipolar organic co-solvent acetonitrile on the observed first-order rate constant (k(obs)) of the pre-steady state acylation phases of the hydrolysis of N-acetyl-Phe-Gly methyl thionester catalysed by the cysteine proteinase variants actinidin and papain in sodium acetate buffer, pH 5.3, were investigated by stopped-flow spectral analysis. With low acetonitrile content, plots of k(obs) against [S]0 for the actinidin reaction are linear with an ordinate intercept of magnitude consistent with a five-step mechanism involving a post-acylation conformational change. Increasing the acetonitrile content results in marked deviations of the plots from linearity with a rate minimum around [S]0=150 microM. The unusual negative dependence of k(obs) on [S]0 in the range 25-150 microM is characteristic of a rate-determining isomerization of the free enzyme before substrate binding, additional to the five-step mechanism. There was no evidence for this phenomenon nor for the post-acylation conformational change in the analogous reaction with papain. For this enzyme, however, acetonitrile acts as an inhibitor with approximately uncompetitive characteristics. Possible mechanistic consequences of the differential solvent-perturbed kinetics are indicated. The free enzyme isomerization of actinidin may provide an explanation for the marked difference in sensitivity between this enzyme and papain of binding site-catalytic site signalling in reactions of substrate-derived 2-pyridyl disulphide reactivity probes.
    Biochemical Journal 04/2004; 378(Pt 2):699-703. · 4.65 Impact Factor
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    ABSTRACT: To investigate the hypothesis that decreased hapten flexibility may lead to increased catalytic antibody activity, we used two closely related immunogens differing only in the flexibility of the atomic framework around the structural motif of the haptens, analogous to the reaction centre of the corresponding substrates. Identical leaving-group determinants in the haptens and identical leaving groups in the substrates removed the ambiguity inherent in some data reported in the literature. Anti-phosphate and anti-phosphonate kinetically homogeneous polyclonal catalytic antibody preparations were compared by using carbonate and ester substrates respectively, each containing a 4-nitrophenolate leaving group. Synthetic routes to a new phosphonate hapten and new ester substrate were developed. The kinetic advantage of the more rigid anti-phosphonate/ester system was demonstrated at pH 8.0 by a 13-fold advantage in k(cat)/k(non-cat) and a 100-fold advantage in the proficiency constant, k(cat)/k (non-cat) x K(m). Despite these differences, the pH-dependences of the kinetic and binding characteristics and the results of chemical modification studies suggest closely similar catalytic mechanisms. The possible origin of the kinetic advantage of the more rigid hapten/substrate system is discussed.
    Biochemical Journal 01/2004; 376(Pt 3):813-21. · 4.65 Impact Factor

Publication Stats

2k Citations
644.67 Total Impact Points

Institutions

  • 1991–2013
    • Queen Mary, University of London
      • School of Biological and Chemical Sciences
      London, ENG, United Kingdom
  • 1975–2011
    • University of London
      • School of Biological Sciences
      Londinium, England, United Kingdom
  • 1999–2010
    • University of Brighton
      • School of Pharmacy and Biomolecular Sciences
      Brighton, England, United Kingdom
  • 2007
    • University of Kent
      • School of Biosciences
      Canterbury, ENG, United Kingdom
  • 1987–2006
    • University of Salford
      Salford, England, United Kingdom
  • 1995
    • The University of York
      York, England, United Kingdom
    • UCL Eastman Dental Institute
      Londinium, England, United Kingdom
  • 1993
    • Dalhousie University
      Halifax, Nova Scotia, Canada
  • 1990–1992
    • Birkbeck, University of London
      Londinium, England, United Kingdom
  • 1989
    • CUNY Graduate Center
      New York City, New York, United States