Mechanism of Benzaldehyde Lyase Studied via Thiamin Diphosphate-Bound Intermediates and Kinetic Isotope Effects †
ABSTRACT Direct spectroscopic observation of thiamin diphosphate-bound intermediates was achieved on the enzyme benzaldehyde lyase, which carries out reversible and highly enantiospecific conversion of ( R)-benzoin to benzaldehyde. The key enamine intermediate could be observed at lambda max 393 nm in the benzoin breakdown direction and in the decarboxylase reaction starting with benzoylformate. With benzaldehyde as substrate, no intermediates could be detected, only formation of benzoin at 314 nm. To probe the rate-limiting step in the direction of ( R)-benzoin synthesis, the (1)H/ (2)H kinetic isotope effect was determined for benzaldehyde labeled at the aldehyde position and found to be small (1.14 +/- 0.03), indicating that ionization of the C2alphaH from C2alpha-hydroxybenzylthiamin diphosphate is not rate limiting. Use of the alternate substrates benzoylformic and phenylpyruvic acids (motivated by the observation that while a carboligase, benzaldehyde lyase could also catalyze the slow decarboxylation of 2-oxo acids) enabled the observation of the substrate-thiamin covalent intermediate via the 1',4'-iminopyrimidine tautomer, characteristic of all intermediates with a tetrahedral C2 substituent on ThDP. The reaction of benzaldehyde lyase with the chromophoric substrate analogue ( E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid and its decarboxylated product ( E)-3-(pyridine-3-yl)acrylaldehyde enabled the detection of covalent adducts with both. Neither adduct underwent further reaction. An important finding of the studies is that all thiamin-related intermediates are in a chiral environment on benzaldehyde lyase as reflected by their circular dichroism signatures.
SourceAvailable from: Xiang Sheng[Show abstract] [Hide abstract]
ABSTRACT: Benzaldehyde lyase (BAL) is a versatile thiamin diphosphate (THDP)-dependent enzyme with widespread synthetic applications in industry. Besides lyase activity, BAL also performs the functions as carboligase and decarboxylase. Unlike many other THDP-dependent enzymes, the active center of BAL is devoid of any acid-base amino acid residues except Glu50 and His29, and therefore, the catalytic mechanism of BAL is unusual. In this article, the dissociation mechanism of (R)-benzoin to benzaldehyde catalyzed by BAL has been studied by using density functional theory method. The calculation results indicate that the whole reaction consists of four elementary steps, and at least two steps contribute to rate-limiting. A big difference with other THDP-dependent enzymes is that, in the first stage of the reaction, the ligation of substrate and THDP ylide is not companied by proton transfer, and in the subsequent transition states and intermediates, the carbonyl oxygen always exists in the form of anion. Gln113, His29, and 4′-amino group of THDP are found to have the function to stabilize the transition states and intermediates. His29 acts as the proton acceptor in step 2 and proton donor in step 3 using one water molecule as mediator. © 2013 Wiley Periodicals, Inc.International Journal of Quantum Chemistry 03/2014; 114(6). DOI:10.1002/qua.24573 · 1.17 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Phosphoketolase (PK) is a thiamine diphosphate (THDP) dependent enzyme which plays key roles in the metabolism of heterofermentative bacteria. By using density functional theory (DFT) method, the catalytic mechanism of PK has been studied on simplified models. The calculation results indicate that the formation of 2-α,β-dihydroxyethylidene-THDP (DHETHDP) and erythrose-4-phosphate (E4P) involves one C–C bond formation and one C–C bond cleavage process. Each C–C bond formation or cleavage is always accompanied by a proton transfer in a concerted but asynchronous way. The dehydration process in the reaction of PK is distinct from that of other THDP-dependent enzymes. The Keto–Enol tautomerism process is assisted with a mediator His553. His64, His553 and His97 are found to have the function to stabilize the transition states and intermediates. His64 is a better candidate of B1 catalyst. His553 acts as a proton donor to protonate the carbonyl oxygen, and plays intermediary role in the Keto–Enol tautomerism process. His97 is the probable B2 catalyst in the dehydration process.Computational and Theoretical Chemistry 12/2013; 1025:1–7. DOI:10.1016/j.comptc.2013.09.026 · 1.37 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Identification of enzyme-bound intermediates via their spectroscopic signatures, which then enable direct monitoring of the kinetic fate of these intermediates, poses a continuing challenge. As an electrophilic covalent catalyst, the thiamin diphosphate (ThDP) coenzyme forms a number of non-covalent and covalent intermediates along its reaction pathways and multiple UV-VIS and circular dichroism (CD) bands have been identified at Rutgers pertinent to several among them. These electronic transitions fall into two classes: those for which the conjugated system provides a reasonable guide to the observed λmax, and others, where there is no corresponding conjugated system, and the observed CD bands are best ascribed to charge transfer (CT) transitions. Herein is reported the reaction of four ThDP enzymes with alternate substrates: (a) acetyl pyruvate, its methyl ester and fluoropyruvate, these providing the shortest side chains attached at the thiazolium C2 atom and leading to CT bands with λmax > 390 nm, not pertinent to any on-pathway conjugated systems (estimated λmax < 330 nm), and (b) (E)-4-(4 chlorophenyl)-2-oxo-3-butenoic acid displaying both a conjugated enamine (430 nm), and a CT transition (480 nm). We suggest that the CT transitions result from an interaction of the π bond on the ThDP C2 side chain as donor, and the positively charged thiazolium ring as acceptor, and correspond to covalent ThDP-bound intermediates. Time resolution of these bands enables determination of rate constants for individual steps. These CD methods are applicable to the entire ThDP superfamily of enzymes and should find applications on other enzymes.Biochemistry 03/2014; 53(13). DOI:10.1021/bi4015743 · 3.19 Impact Factor