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The mechanism of the enzyme benzoylformate decarboxylase (BFDC), which carries out a typical thiamin diphosphate (ThDP)-dependent nonoxidative decarboxylation reaction, was studied with the chromophoric alternate substrate (E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid (3-PKB). Addition of 3-PKB resulted in the appearance of two transient intermediates formed consecutively, the first one to be formed a predecarboxylation ThDP-bound intermediate with lambda(max) at 477 nm, and the second one corresponding to the first postdecarboxylation intermediate the enamine with lambda(max) at 437 nm. The time course of formation/depletion of the PKB-ThDP covalent complex and of the enamine showed that decarboxylation was slower than formation of the PKB-ThDP covalent adduct. When the product of decarboxylation 3-(pyridin-3-yl)acrylaldehyde (PAA) was added to BFDC, again an absorbance with lambda(max) at 473 nm was formed, corresponding to the tetrahedral adduct of PAA with ThDP. Addition of well-formed crystals of BFDC to a solution of PAA resulted in a high resolution (1.34 A) structure of the BFDC-bound adduct of ThDP with PAA confirming the tetrahedral nature at the C2alpha atom, rather than of the enamine, and supporting the assignment of the lambda(max) at 473 nm to the PAA-ThDP adduct. The structure of the PAA-ThDP covalent complex is the first example of a product-ThDP adduct on BFDC. Similar studies with 3-PKB indicated that decarboxylation had taken place. Evidence was also obtained for the slow formation of the enamine intermediate when BFDC was incubated with benzaldehyde, the product of the decarboxylation reaction thus confirming its presence on the reaction pathway.

The mandelate pathway of Pseudomonas putida ATCC 12633 comprises five enzymes and catalyzes the conversion of R - and S -mandelamide to benzoic acid which subsequently enters the β-ketoadipate pathway. Although the first four enzymes have been extensively characterized the terminal enzyme, a NAD(P) + -dependent benzaldehyde dehydrogenase (BADH), remains largely undescribed. Here we report that BADH is a dimer in solution, and that DTT is necessary both to maintain the activity of BADH and to prevent oligimerization of the enzyme. Site-directed mutagenesis confirms that Cys249 is the catalytic cysteine and identifies Cys140 as the cysteine likely to be involved in inter-monomer disulfide formation. BADH can utilize a range of aromatic substrates and will also operate efficiently with cyclohexanal as well as medium-chain aliphatic aldehydes. The log V and log V / K pH-rate profiles for benzaldehyde with either NAD + or NADP + as the coenzyme are both bell-shaped. The p K a values on the ascending limb range from 6.2 to 7.1 while those on the descending limb range from 9.6 to 9.9. A spectrophotometric approach was used to show that the p K a of Cys249 was 8.4, i.e., Cys249 is not responsible for the p K a s observed in the pH-rate profiles.

Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of ( R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg (2+) as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these types of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analogue of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 A (Protein Data Bank entry 3D7K ) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase.

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.

Plasmodium falciparum thioredoxin reductase (PfTrxR: NADPH + Trx(S)2 + H+ ↔ NADP+ + Trx(SH)2) is a high Mr flavin-dependent TrxR that reduces thioredoxin (Trx) via a CysXXXXCys pair located penultimately to the C-terminal Gly. In this respect, PfTrxR differs significantly from its human counterpart which bears a Cys-Sec redox pair at the same position. PfTrxR is essentially involved in antioxidant defense and redox regulation of the parasite and has been previously validated by knock-out studies as a potential drug target for malaria chemotherapy. Moreover, human TrxR is present in most cancer cells at levels tenfold higher than in normal cells. Here we report the discovery of a series of potent inhibitors of PfTrxR. The three most promising inhibitors, 3 ( IC 50 PfTrxR = 2 μ M and IC 50 hTrxR = 50 μ M ) , 7 ( IC 50 PfTrxR = 2 μ M and IC 50 hTrxR = 140 μ M ) , and 11 ( IC 50 PfTrxR = 0.5 μ M and IC 50 hTrxR = 4 μ M ) were selective for the parasite enzyme. Detailed mechanistic characterization of the effects of these compounds on the PfTrxR-catalyzed reaction showed clear uncompetitive inhibition with respect to both substrate and cofactor. For the most specific PfTrxR inhibitor 7, an alkylation mechanism study based on a thiol conjugation model was performed. Furthermore, all three compounds were active in the lower micromolar range on the chloroquine-resistant P. falciparum strain K1 in vitro.

Found in all vertebrates, creatine kinase catalyzes the reversible reaction of creatine and ATP forming phosphocreatine and ADP. Phosphocreatine may be viewed as a reservoir of "high-energy phosphate" which is able to supply ATP, the primary energy source in bioenergetics, on demand. Consequently, creatine kinase plays a significant role in energy homeostasis of cells with intermittently high energy requirements. The enzyme is of clinical importance and its levels are routinely used as an indicator of myocardial and skeletal muscle disorders and for the diagnosis of acute myocardial infarction. First identified in 1928, the enzyme has undergone intensive investigation for over 75 years. There are four major isozymes, two cytosolic and two mitochondrial, which form dimers and octamers, respectively. Depending on the pH, the enzyme operates by a random or an ordered bimolecular mechanism, with the equilibrium lying towards phosphocreatine production. Evidence suggests that conversion of creatine to phosphocreatine occurs via the in-line transfer of a phosphoryl group from ATP. A recent X-ray structure of creatine kinase bound to a transition state analog complex confirmed many of the predictions based on kinetic, spectroscopic, and mutagenesis studies. This review summarizes and correlates the more significant mechanistic and structural studies on creatine kinase.

2-Naphthalenesulfonic acid (4-hydroxy-7-[[[[5-hydroxy-6-[(4 cinnamylphenyl)azo]-7-sulfo-2-naphthalenyl]amino]-carbonyl]amino]-3-[(4-cinnamylphenyl)]azo (KM-1)) is a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) that was designed to bind at an unconventional site on human immunodeficiency virus type 1 reverse transcriptase (RT) (Skillman, A. G., Maurer, K. W., Roe, D. C., Stauber, M. J., Eargle, D., Ewing, T. J., Muscate, A., Davioud-Charvet, E., Medaglia, M. V., Fisher, R. J., Arnold, E., Gao, H. Q., Buckheit, R., Boyer, P. L., Hughes, S. H., Kuntz, I. D., and Kenyon, G. L. (2002) Bioorg. Chem. 30, 443–458). We have investigated the mechanism by which KM-1 inhibits wild-type human immunodeficiency virus type 1 RT by using pre-steady state kinetic methods to examine the effect of KM-1 on the parameters governing the single nucleotide incorporation catalyzed by RT. Analysis of the pre-steady-state burst phase of dATP incorporation showed that KM-1 decreased the amplitude of the reaction as previously shown for other NNRTIs, because of the slow equilibration of the inhibitor with RT. In the ternary enzyme-DNA-KM-1 complex (E-DNA-I), incorporation of the next nucleotide onto the primer is blocked. However, unlike conventional NNRTIs, the inhibitory effect was caused primarily by weakening the DNA binding affinity and displacing DNA from the enzyme. Wild-type RT binds a 25/45-mer DNA duplex with an apparent Kd of 3 nm, which was increased to 400 nm upon saturation with KM-1. Likewise, the apparent Kd for KM-1 binding to RT increased at higher DNA concentrations. We therefore conclude that KM-1 represents a new class of inhibitor distinct from nevirapine and related NNRTIs. KM-1 can bind to RT in both the absence and presence of DNA but weakens the affinity for DNA 140-fold so that it favors DNA dissociation. The data suggest that KM-1 distorts RT conformation and misaligns DNA at the active site.

Benzoylformate decarboxylase is a member of the family of enzymes that are dependent on the cofactor thiamin diphosphate. A structure of this enzyme binding (R)-mandelate, a competitive inhibitor, suggests that at least two hydrogen bonds are formed between the substrate, benzoylformate, and active site side chains. The first is between the carboxylate group of benzoylformate and the hydroxyl group of S26, and the second is between carbonyl group of the substrate and an imidazole nitrogen of H70. Steady-state kinetic studies indicate that the catalytic parameters are strongly affected in three active site mutants, S26A, H70A, and H281A. The K(m) of S26A was increased most dramatically, 25-fold more than that of the wild-type enzyme, while the K(i) of (R)-mandelate was increased 100-fold, suggesting that the serine hydroxyl is important for substrate binding. The k(cat) of H70A is reduced more than 3 orders of magnitude, strongly implicating this residue in catalysis, and H281 showed significant, but smaller magnitude, effects on both K(m) and k(cat). Stopped-flow experiments using an alternative substrate, p-nitrobenzoylformate, lead to kinetic resolution of the fate of key thiamin diphosphate-bound intermediates. Together, the experimental results suggest the following roles for residues in the active site. The residue H70 is important for the protonation of the 2-alpha-mandelyl-ThDP intermediate, thereby assisting in decarboxylation, and for the deprotonation of the 2-alpha-hydroxybenzyl-ThDP intermediate, aiding product release. H281 is involved in protonation of the enamine. Surprisingly, S26 appears to be involved not only in substrate binding but also in other steps of the reaction.

Selective inhibitors of enzyme-catalyzed reactions are widely used in both biochemical and medical science. The inhibitor may be used to block either a single enzyme or a metabolic pathway. The utility of an enzyme inhibitor as a mechanistic probe or a therapeutic agent will depend, in part, on the potency of the inhibitor and its specificity toward its target enzyme. These properties will, in turn, depend on the number and type of interactions the inhibitor makes with the enzyme and the overall mode of inhibition. Here we provide a concise overview of the forces involved in inhibitor binding, and a brief summary of the treatment of enzyme kinetic data. Enzyme inhibitors have been divided into two groups, reversible and irreversible, based on whether the inhibitor binds covalently to its target enzyme. Within these groups inhibitors have been classified on the basis of kinetics, structure, and mechanism. Examples are provided in all cases and, where possible, these are of inhibitors possessing therapeutic interest. The criteria for inclusion in each class of inhibitor are described, as well as the kinetic implications, with particular reference to the specificity of the inhibition. Treatment of the examples includes discussion of the design aspects and, if appropriate, a historical perspective that demonstrates the development of a rational approach to inhibitor design. Keywords: enzyme inhibitor; inhibition constant; IC50 value; multisubstrate analog; transition state analog; affinity label; mechanism-based inhibitor

The human immunodeficiency virus (HIV) epidemic is an important medical problem. Although combination drug regimens have produced dramatic decreases in viral load, current therapies do not provide a cure for HIV infection. We have used structure-based design and combinatorial medicinal chemistry to identify potent and selective HIV-1 reverse transcriptase (RT) inhibitors that may work by a mechanism distinct from that of current HIV drugs. The most potent of these compounds (compound 4 , 2-naphthalenesulfonic acid, 4-hydroxy-7-[[[[5-hydroxy-6-[(4-cinnamylphenyl)azo]-7-sulfo-2-naphthalenyl]amino]carbonyl]amino]-3-[(4-cinnamylphenyl)azo], disodium salt) has an IC 50 of 90 nM for inhibition of polymerase chain extension, a K d of 40 nM for inhibition of DNA–RT binding, and an IC 50 of 25–100 nM for inhibition of RNaseH cleavage. The parent compound ( 1 ) was as effective against 10 nucleoside and non-nucleoside resistant HIV-1 RT mutants as it was against the wild-type enzyme. Compound 4 inhibited HIV-1 RT and murine leukemia virus (MLV) RT, but it did not inhibit T 4 DNA polymerase, T 7 DNA polymerase, or the Klenow fragment at concentrations up to 200 nM. Finally, compound 4 protected cells from HIV-1 infection at a concentration more than 40 times lower than the concentration at which it caused cellular toxicity.

Creatine kinase (CK) catalyzes the reversible conversion of creatine and ATP to phosphocreatine and ADP, thereby helping maintain energy homeostasis in the cell. Here we report the first X-ray structure of CK bound to a transition-state analogue complex (CK-TSAC). Cocrystallization of the enzyme from Torpedo californica (TcCK) with ADP-Mg(2+), nitrate, and creatine yielded a homodimer, one monomer of which was liganded to a TSAC complex while the second monomer was bound to ADP-Mg(2+) alone. The structures of both monomers were determined to 2.1 A resolution. The creatine is located with the guanidino nitrogen cis to the methyl group positioned to perform in-line attack at the gamma-phosphate of ATP-Mg(2+), while the ADP-Mg(2+) is in a conformation similar to that found in the TSAC-bound structure of the homologue arginine kinase (AK). Three ligands to Mg(2+) are contributed by ADP and nitrate and three by ordered water molecules. The most striking difference between the substrate-bound and TSAC-bound structures is the movement of two loops, comprising residues 60-70 and residues 323-332. In the TSAC-bound structure, both loops move into the active site, resulting in the positioning of two hydrophobic residues (one from each loop), Ile69 and Val325, near the methyl group of creatine. This apparently provides a specificity pocket for optimal creatine binding as this interaction is missing in the AK structure. In addition, the active site of the transition-state analogue complex is completely occluded from solvent, unlike the ADP-Mg(2+)-bound monomer and the unliganded structures reported previously.

The pET17 expression vector was used to express creatine kinase from the electric organ of Torpedo californica as inclusion bodies in Escherichia coli BL21(DE3) cells. The insoluble aggregate was dissolved in 8 M urea and, following extraction with Triton X-100, the enzyme was refolded by dialysis against Tris buffer (pH 8.0) containing 0.2 M NaCl. After two buffer changes, chromatography on Blue Sepharose was used as a final step in the purification procedure. Approximately 54 mg active protein was recovered from a 1 L culture and the refolded enzyme had a specific activity of 75 U/mg. The molecular mass of the purified protein was consistent with that predicted from the amino acid sequence and the CD spectrum of the refolded enzyme was essentially identical to that of creatine kinase from human muscle (HMCK). The K m values of ATP and ADP were also similar to those of HMCK, while the K m values for both phosphocreatine and creatine were approximately 5–10-fold higher. The purification described here is in marked contrast with earlier attempts at purification of this isozyme where, in a process yielding less than 1 mg/L culture, enzyme with a specific activity of ca. 5 U/mg was obtained.

Thiamin diphosphate (ThDP)-dependent enzymes catalyze a range of transformations, such as decarboxylation and ligation. We report a novel spectroscopic assay for detection of some of the ThDP-bound intermediates produced on benzoylformate decarboxylase. Benzoylformate decarboxylase was mixed with its alternate substrate p-nitrobenzoylformic acid on a rapid-scan stopped-flow instrument, resulting in formation of three absorbing species (lambda(max) in parentheses): I(1) (a transient at 620 nm), I(2) (a transient at 400 nm), and I(3) (a stable absorbance with lambda(max)730 nm). Analysis of the kinetics of the two transient species supports a model in which a noncovalent complex of the substrate and the enzyme is converted to the first covalent intermediate I(1); the absorbance corresponding to I(1) is probably a charge-transfer band arising from the interaction of the thiamin diphosphate-p-nitrobenzoylformic acid covalent adduct (2-p-nitromandelylThDP) and the enzyme. The rate of disappearance of I(1) parallels the rate of formation of I(2). Chemical models suggest the lambda(max) of I(2) (near 400 nm) to be appropriate to the enamine, a key intermediate in ThDP-dependent reactions resulting from the decarboxylation of the thiamin diphosphate-p-nitrobenzoylformic acid covalent adduct. Therefore, the rate of disappearance of I(1) and/or the appearance of I(2) directly measure the rate of decarboxylation. A relaxation kinetic treatment of the pre-steady-state kinetic data also revealed a hitherto unreported facet of the mechanism, alternating active-sites reactivity. Parallel studies of the His70Ala BFD active-site variant indicate that it cannot form the complex reported by the charge-transfer band (I(1)) at the level of the wild-type protein.

BACKGROUND: Protozoan parasites of the genus Trypanosoma cause disease in a wide range of mammalian hosts. Trypanosoma brucei brucei, transmitted by tsetse fly to cattle, causes a disease (Nagana) of great economic importance in parts of Africa. T. b. brucei also serves as a model for related Trypanosoma species, which cause human sleeping sickness. MATERIALS AND METHODS: Chalcone and acyl hydrazide derivatives are known to retard the growth of Plasmodium falciparum in vitro and inhibit the malarial cysteine proteinase, falcipain. We tested the effects of these compounds on the growth of bloodstream forms of T. b. brucei in cell culture and in a murine trypanosomiasis model, and investigated their ability to inhibit trypanopain-Tb, the major cysteine proteinase of T. b. brucei. RESULTS: Several related chalcones, acyl hydrazides, and amides killed cultured bloodstream forms of T. b. brucei, with the most effective compound reducing parasite numbers by 50% relative to control populations at a concentration of 240 nM. The most effective inhibitors protected mice from an otherwise lethal T. b. brucei infection in an in vivo model of acute parasite infection. Many of the compounds also inhibited trypanopain-Tb, with the most effective inhibitor having a Ki value of 27 nM. Ki values for trypanopain-Tb inhibition were up to 50- to 100-fold lower than for inhibition of mammalian cathepsin L, suggesting the possibility of selective inhibition of the parasite enzyme. CONCLUSIONS: Chalcones, acyl hydrazides, and amides show promise as antitrypanosomal chemotherapeutic agents, with trypanopain-Tb possibly being one of their in vivo targets.

We report the expression of the human muscle (CK-MM) and brain (CK-BB) creatine kinases in Escherichia coli. The proteins have been purified to apparent homogeneity and several of their physical and kinetic properties investigated. In the process, we have conclusively verified the correct DNA sequence of the genes encoding the respective isozymes, and determined the correct primary structure and mass of the gene products. Alignment of the primary sequences of these two enzymes shows 81% sequence identity with each other, and no obvious gross structural differences. However, Western blot analyses demonstrated the general lack of antigenic cross-reactivity between these isozymes. Preliminary kinetic analyses show the K(m) and k(cat) values for the creatine and MgATP substrates are similar to values reported for other isozymes from various tissues and organisms. The human muscle and brain CKs do not, however, exhibit the synergism of substrate binding that is observed, for example, in rabbit muscle creatine kinase.

The accumulated evidence concerning the catalytic role of rabbit muscle creatine kinase will be discussed in light of the recently reported x-ray structures of an arginine kinase and a creatine kinase from chicken mitochondrion.

We have developed and applied a computational strategy to increase the affinity of fullerene-based inhibitors of the HIV protease. The result is a approximately 50-fold increase in affinity from previously tested fullerene compounds. The strategy is based on the design of derivatives which may potentially increase hydrophobic desolvation upon complex formation, followed by the docking of the hypothetical derivatives into the HIV protease active site and assessment of the model complexes so formed. The model complexes are generated by the program DOCK and then analyzed for desolvated hydrophobic surface. The amount of hydrophobic surface desolvated was compared with a previously tested compound, and if this amount was significantly greater, it was selected as a target. Using this approach, two targets were identified and synthesized, using two different synthetic approaches: a diphenyl C60 alcohol (5) based on a cyclopropyl derivative of Bingel (Chem.Ber. 1993, 126, 1957-1959) and a diisopropyl cyclohexyl C60 alcohol (4a) as synthesized by Ganapathi et al. (J. Org.Chem. 1995, 60, 2954-2955). Both showed tighter binding than the originally tested compound (diphenethylaminosuccinate methano-C60, Ki = 5 microM) with Ki values of 103 and 150 nM, respectively. In addition to demonstrating the utility of this approach, it shows that simple modification of fullerenes can result in high-affinity ligands of the HIV protease, for which they are highly complementary in structure and chemical nature.

Solid support-bound chemistry has been used to gain access to several polyfunctional ureas which could not be easily produced via traditional solution phase approaches.

Six deoxynucleoside triphosphate (dNTP) analogues were synthesized, having the α,β-P-O-P bond replaced with an imido (P-N-P) functionality. They were all shown to be reasonably potent inhibitors of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT). This has permitted, for the first time, an estimate of the relative binding affinities of the parent triphosphates (dATP, TTP, dGTP, and dCTP) toward the enzyme's active site, in that they can be compared indirectly by correlation with the behavior of their α,β-imido analogues. Other complicating processes such as consecutive incorporation into the growing DNA chain can be excluded because the imido linkage cannot be cleaved by HIV-1 RT. The 5-iodo analog of deoxyuridine triphosphate (IdUMPNPP, 5 ) was the most potent inhibitor, having an IC 50 value of 7 μ M . A general route for the phosphorylation of purine and pyrimidine 5′-imidodiphosphates by pyruvate kinase was developed using phosphoenolpyruvate (PEP) as a phosphoryl group donor. Enzymatic phosphorylation was shown to be a more efficient approach than chemical methods.

The first solid-phase synthesis of novel peptide-derived enantiospecific nucleic acid analogs (PDNAs), employing all three stereoisomers of 4-( N 9 -adeninyl)-2-amino( t -boc)-butyric acid as ADNA (amino acid-derived nucleoside analogs) monomers has been described. The preliminary melting temperature ( T m ) data obtained by hybridization experiments demonstrated that these PDNAs can anneal to their complementary nucleic acid strand dT 10 .

A convergent and cost-effective strategy to synthesize enantiospecific nucleoside/ nucleotide analogs from readily available α-amino acids has been described. Both ( L )- and ( D )-methionine were transformed in four steps to the corresponding key intermediates ( L )- and ( D )- 2-(tert-butyloxycarbonylamino)-4-bromomethyl butyrate. Nucleophilic displacement of the bromide by nucleic acid bases (e.g., adenine, thymine, guanine, cytosine) provided the enantiomerically pure monomers required for the solid-phase synthesis of novel peptide-derived enantiospecific nucleic acid analogs.

Creatine kinase (CK; EC 2.7.3.2) catalyzes the reversible conversion of creatine and MgATP to phosphocreatine and MgADP. In the absence of an X-ray crystal structure, we have used the sequence homology of creatine kinases and other guanidino kinases from a variety of sources to identify the conserved histidine residues in rabbit muscle CK, as well as to try to pinpoint a reactive histidine that has been implicated in the active site. This residue has been proposed to act as a general acid/base catalyst assisting in the phosphoryl transfer mechanism [Cook et al. (1981) Biochemistry 20, 1204-1210]. There are 17 histidine residues in rabbit muscle CK, and of these, only five have been conserved in all guanidino kinase sequences published to date [Mühlebach et al. (1994) Mol. Cell. Biochem. 133, 245-62]. In rabbit muscle CK, these residues are H96, H105, H190, H233, and H295. We have carried out site-specific mutagenesis of these five histidine residues, replacing each with an asparagine. Each of these mutants exhibited enzymatic activity but to varying degrees. The H105N, H190N, and H233N mutants displayed specific activities similar to that of the wild-type enzyme. H96N has high activity, but appears to be quite unstable, losing catalytic activity upon cell lysis by sonication and/or chromatographic steps involved in purification. H295N shows a significantly reduced catalytic activity relative to the native enzyme, due to marked decreases in kcat and the affinities for both substrates. Each of the five mutants is inactivated by diethyl pyrocarbonate (DEP), and inactivation is reversible upon incubation with hydroxylamine. However, only H295N shows a dramatically reduced rate of inactivation relative to native CK, consistent with H295 being the residue modified by DEP in the native enzyme. These intriguing results indicate that four of the conserved histidines (H96, H105, H295, and H233) are not essential for activity, and while H295 may be at the active site of CK, it is unlikely to play the role of a general acid/base catalyst.

We have discovered a superfamily of enzymes related by their ability to catalyze the abstraction of the alpha-proton of a carboxylic acid to form an enolic intermediate. Although each reaction catalyzed by these enzymes is initiated by this common step, their overall reactions (including racemization, beta-elimination of water, beta-elimination of ammonia, and cycloisomerization) as well as the stereochemical consequences (syn vs anti) of the beta-elimination reactions are diverse. Analysis of sequence and structural similarities among these proteins suggests that all of their chemical reactions are mediated by a common active site architecture modified through evolution to allow the enolic intermediates to partition to different products in their respective active sites via different overall mechanisms. All of these enzymes retain the ability to catalyze the thermodynamically difficult step of proton abstraction. These homologous proteins, designated the "enolase superfamily", include enolase as well as more metabolically specialized enzymes: mandelate racemase, galactonate dehydratase, glucarate dehydratase, muconate-lactonizing enzymes, N-acylamino acid racemase, beta-methylaspartate ammonia-lyase, and o-succinylbenzoate synthase. Comparative analysis of structure-function relationships within the superfamily suggests that carboxyphosphonoenolpyruvate synthase, another member of the superfamily, does not catalyze the reaction proposed in the literature but catalyzes an enolase-like reaction instead. The established and deduced structure-function relationships in the superfamily allow the prediction that other apparent members of the family for which no catalytic functions have yet been assigned will also perform chemistry involving abstraction of the alpha-protons of carboxylic acids.

Acetoacetate decarboxylase from Clostridium acetobutylicum (AAD) catalyzes the decarboxylation of acetoacetate via a Schiff base intermediate [Hamilton, G. A., & Westheimer, F. H. (1959) J. Am. Chem. Soc. 81, 6332; Fridovich, I., & Westheimer F. H. (1962) J. Am. Chem. Soc. 84, 3208]. The pKa of the active-site lysine (Lys 115) is 6.0, 4.5 pKa units less than the pKa of lysine in solution [Kokesh, F. C., & Westheimer, F. H. (1971) J. Am. Chem. Soc. 93, 7270; Frey, P. A., Kokesh, F. C., & Westheimer, F. H. (1971) J. Am. Chem. Soc. 93, 7266; Schmidt, D. E., Jr., & Westheimer, F. H. (1971) Biochemistry 10, 1249]. Westheimer and co-workers hypothesized that the pKa of Lys 115 is decreased by its spatial proximity to the epsilon-ammonium group of Lys 116. We have investigated this proposal by studying site-directed mutants of Lys 115 and Lys 116. Two substitutions for Lys 115 (K115C and K115Q) were both catalytically inactive at pH 5.95, the pH optimum of wild type AAD, demonstrating the importance of this residue in catalysis. Activity could be restored to K115C by aminoethylation with 2-bromoethyl-ammonium bromide (2-BEAB). Substitutions for Lys 116 (K116C, K116N, and K116R) had reduced but significant activities at pH 5.95. The effects of Lys 116 on the pKa of Lys 115 in the mutant AADs were evaluated following imine formation with 5-nitrosalicylaldehyde and reduction with NaBH4. Whereas the pKa of Lys 115 in K116R is similar to that observed for wild type AAD, the pKaS of Lys 115 in K116C and K116N were elevated to > 9.2. Alkylation of Cys 116 in K116C with 2-BEAB resulted in both significant activation and restoration of the pKa of Lys 115 to 5.9. These data support Westheimer's hypothesis that the pKa of the Schiff base-forming Lys 115 is decreased by its spatial proximity to the epsilon-ammonium group of Lys 116.

Isoelectric focusing separation of recombinant rabbit muscle creatine kinase (CK) and its 282Cys-->282Ser mutant shows the presence of three and two isoforms, respectively, that exhibit equivalent enzymatic activity. Electrospray ionization coupled with Fourier-transform mass spectrometry (10(5) resolving power) of both CKs indicates that their major components are within +/- 2 Da of the M(r) value predicted from the cDNA sequences of these mixtures. Dissociation of (M + nH)n+ gives no evidence that the components of either CK are isomers; the masses of the 51 fragment ions correlate completely (+/- 1 Da) with the values predicted from the cDNA sequence and confirm the identities of 21 of the 380 amino acids and the 282Cys-->282Ser replacement in the mutant. The results are consistent with one or two steps of post-translational amidation/deamidation (NH2-->OH, 16 Da-->17 Da), each of which would produce only a 1 Da difference in M(r), with the fragment masses indicating that at least one modification occurs between residues 212 and 282.

A series of chalcones and their derivatives have been synthesized and identified as novel potential antimalarials using both molecular modeling and in vitro testing against the intact parasite. A large number of chalcones and their derivatives were prepared using one-step Claisen-Schmidt condensations of aldehydes with methyl ketones. These condensates were screened in vitro against both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum and shown to be active at concentrations in the nanomolar range. The most active chalcone derivative, 1-(2,5-dichlorophenyl)-3-(4-quinolinyl)-2-propen-1-one (7), had an IC50 value of 200 nM against both a chloroquine-resistant strain (W2) and a chloroquine-sensitive strain (D6). The resistance indexes for all compounds were substantially lower than for chloroquine, suggesting that this series will be active against chloroquine-resistant malaria. Structure-activity relationships (SAR) of the chalcones in the context of a homology-based model structure of the malaria trophozoite cysteine protease, the most likely target enzyme, are presented.

A simple, rapid, and sensitive spectrophotometric method has been developed for the determination of alpha-keto acids, alpha-diketones, and alpha-ketoaldehydes using o-phenylenediamine as a derivatizing reagent in acidic media to yield chromophoric quinoxaline derivatives. Application to the determination of alpha-ketobutyrate and the rate of its formation from isomerization of vinylglycolate catalyzed by mandelate racemase is described. Under optimal conditions 1 to 4 mM o-phenylenediamine in 80% acetic acid was incubated at 50 degrees C for 10 min with the alpha-dicarbonyl compounds. Solutions of alpha-ketobutyrate as low as 5 microM in the buffer solution can be measured quantitatively. This method has also been used for determining the rate of formation of the phenylglyoxalate from (S)-mandelate catalyzed by (S)-mandelate dehydrogenase.

A novel property of the bacterial outer membrane protein T, OmpT, has been discovered. It is active under extreme denaturing conditions. This finding emerged during characterization of a protease associated with the degradation of recombinant proteins expressed as inclusion bodies in Escherichia coli. These inclusion body proteins are stable to proteolytic degradation until they are solubilized by denaturation. The protease that degrades them under denaturing conditions was identified as OmpT on the basis of substrate specificity, inhibitor profile, and confirmation that its N-terminal sequence is identical with that of OmpT. A previously unknown property of this enzyme, OmpT's preference for denatured substrates, may provide a clue to its physiological function. To facilitate further characterization of this proteolytic activity, we have optimized a system to extract and assay OmpT under denaturing conditions using a soluble substrate, rabbit muscle creatine kinase.

Background : The trophozoite stage of the malaria parasite infects red blood cells. During this phase of their lifecycle, the parasites use hemoglobin as their principal source of amino acids, using a cysteine protease to degrade it. We have previously reported a three-dimensional model of this cysteine protease, based on the structures of homologous proteases, and the use of the program DOCK to identify a ligand for the malaria protease. Results : Here we describe the design of improved ligands starting from this lead. Ligand design was based on the predicted configuration of the lead compound docked to the model three-dimensional structure of the protease. The lead compound has an IC 50 of 6 μM, and our design/synthesis strategy has resulted in increasingly potent derivatives that block the ability of the parasites to infect and/or mature in red blood cells. The two best derivatives to date have IC 50 s of 450 nM and 150 nM. Conclusions : A new class of anti-malarial chemotherapeutics has resulted from a computational search that was based on a model of the target protease. Despite the lack of a detailed experimental structure of the target enzyme or the enzyme-inhibitor complex, we have been able to identify compounds with increased potency. These compounds approach the activity of chloroquine (IC 50 = 20 nM), but have a distinct mechanism of action. This series of compounds could thus lead to new therapies for chloroquine-resistant malaria.

The three-dimensional solution structure of a trisdecamer DNA duplex sequence, d(AGCTTGCCTTGAG).d(CTCAAGGCAAGCT), from a conserved region of HIV-1 genome's long terminal repeat, has been investigated using NMR spectroscopy and restrained molecular dynamics calculations. Interproton distances derived from two-dimensional nuclear Overhauser enhancement (2D NOE) experiments, using the iterative complete relaxation matrix algorithm MARDIGRAS, and torsion angles for sugar rings, estimated from stimulated fitting of double-quantum-filtered correlation (2QF-COSY) spectra, were obtained [Mujeeb, A., Kerwin, S. M., Egan, W. M., Kenyon, G. L., & James, T. L. (1992) Biochemistry 31, 9325-38]. These structural restraints have now been employed as the basis for structure refinement using restrained molecular dynamics (rMD) to search conformational space for structures consistent with the experimental restraints. Specifically, upper and lower bounds on the restraints were incorporated into the AMBER (version 4.0) total potential energy function of the system, the bounds being used to define the width of a flat-well penalty term in the AMBER force field. Confidence in the time-averaged structure obtained is engendered by convergence to essentially the same structure (root-mean-square deviation approximately 0.9 A) when two quite different DNA models, A-DNA and B-DNA (RMSD approximately 6.5 A), were employed as starting structures and when various initial trajectories were used for the rMD runs. The derived structure is further supported by the total energy calculated, the restraint violation energy, the restraint deviations, and the fit with experimental data. For the latter, the sixth-root residual index indicated a good fit of the determined structure with experimental 2D NOE spectral intensities (R1x < 0.07), and the RMS difference between vicinal proton coupling constants calculated for the derived structure and experimental coupling constants were also in reasonable agreement (JRMS = 0.9 Hz). While the structure of the trisdecamer is basically in the B-DNA family, some structural parameters manifest interesting local variations. The helix parameters indicate that, compared with classical B-DNA, the structure is longitudinally more compressed. Local structural variations at the two TG steps in particular together create bending into the major groove of the duplex. Comparison of the two-CTTG-tetrads in the duplex reveals that they have similar structures, with the TT moieties being almost identical; however, the -CTTG-pur sequence has a larger roll and slide for the -TG- step than for the -CTTG-pyr sequence, in accord with published X-ray crystallographic conclusions.

The (S)-mandelate dehydrogenase (MDH) from Pseudomonas putida (ATCC 12633) is the only membrane-associated member of a homologous family of FMN-dependent, alpha-hydroxy acid dehydrogenases/oxidases that includes the structurally characterized glycolate oxidase from spinach (GOX). We have correlated the membrane association of MDH to a polypeptide segment in the interior of the primary sequence. This has been accomplished by construction of a chimeric enzyme in which the putative membrane-binding segment in MDH has been deleted and replaced with the corresponding segment from the soluble GOX. The resulting chimera, MDH-GOX, is soluble and retains partial catalytic activity (approximately 1%) using (S)-mandelate as substrate. In contrast, the activities of both the membrane-associated wild-type MDH and the soluble MDH-GOX are nearly the same when (S)-phenyllactate is used as substrate. To the best of our knowledge, this is the first example of a membrane-associated protein in which an internal polypeptide segment anchors the protein to the membrane.

The ability of C[sub 60] fullerene ([open quotes]Bucky Ball[close quotes]) derivatives to interact with the active site of HIV-1 protease (HIVP) has been examined through model building and simple physical chemical analysis. The model complexes generated via the program DOCK3 suggest that C[sub 60] derivatives will fit snugly in the active site, thereby removing 298 A[sup 2] of primarily nonpolar surface from solvent exposure and driving ligand/protein association. The prediction that these compounds should bind to the active site and thereby act as inhibitors has been borne out by the experimental evidence. Kinetic analysis of HIVP in the presence of a water-soluble C[sub 60] derivative, bis(phenethylamino-succinate) C[sub 60], suggests a competitive mode of inhibition. This is consistent with and supports the predicted binding mode. Diamino C[sub 60] has been proposed as a [open quotes]second-generation[close quotes] C[sub 60] derivative that will be able to form salt bridges with the catalytic aspartic acids in addition to Van der Waals contacts with the nonpolar HIVP surface, thereby improving the binding relative to the tested compound. 15 refs., 6 figs., 1 tab.

A diamido diacid diphenyl fulleroid derivative was designed specifically to inhibit an HIV enzyme. The detailed synthesis and mass spectrometric analysis of the water-soluble, biologically active methanofullerene are described. The compound was prepared in three steps from C[sub 60] via a suitably diphenyldiazomethane. High-resolution mass spectrometric analysis was possible only under mild matrix-assisted laser desorption/ionization Fourier transform mass spectrometry conditions. Direct infrared or ultraviolet laser desorption resulted exclusively in observation of C[sub 60] ions, in either positive or negative mode. 13 refs., 3 figs.

A series of chalcones and their derivatives have been synthesized and identified as novel potential antimalarials using both molecular modeling and in vitro testing against the intact parasite. A large number of chalcones and their derivatives were prepared using one-step Claisen-Schmidt condensations of aldehydes with methyl ketones. These condensates were screened in vitro against both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum and shown to be active at concentrations in the nanomolar range. The most active chalcone derivative, 1-(2,5-dichlorophenyl)-3-(4-quinolinyl)-2-propen-1-one (7), had an IC50 value of 200 nM against both a chloroquine-resistant strain (W2) and a chloroquine-sensitive strain (D6). The resistance indexes for all compounds were substantially lower than for chloroquine, suggesting that this series will be active against chloroquine-resistant malaria. Structure-activity relationships (SAR) of the chalcones in the context of a homology-based model structure of the malaria trophozoite cysteine protease, the most likely target enzyme, are presented.

Propargylglycolate (2-hydroxy-3-butynoic acid) has been determined to be both a substrate and an inactivator of mandelate racemase. The partition ratio for racemization/inactivation has been estimated to be ∼17,000. Inactivation of the racemase appears to require the rapid covalent addition of 1 substrate molecule; however, a slower labeling process is observed that results in the attachment of up to 5 molecules of substrate per active site. The process is consistent with an enzyme-catalyzed rearrangement of the acetylenic substrate to an allenic-enol that affords 2-keto-3-butenoate as the ultimate electrophile.

Mandalate racemase and class-related enzymes have attracted much interest as potentially simple models for studying the chemical basis of enzymic catalysis. Recent studies on mandelate racemase have been particularly fruitful, revealing the structural workings of a racemase for the first time.

The importance of creatine kinase (E.C. 2.7.3.2) in endocrine tissues has been generally overlooked. Using a specific radiometric assay, we have demonstrated the existence of CK in the Brockmann body (principal islet) of the Coho salmon. We have purified this protein from insular tissue and concurrently purified CK from brain and muscle of the salmon. Purification characteristics, immunological cross-reactivity, and N-terminal sequence analysis have demonstrated that the predominant cytosolic CK from the Brockmann body is indistinguishable from the BB (brain) isoenzyme. Immunocytochemical studies indicated that the enzyme resides in the endocrine parenchyma. Phosphocreatine may serve as a reservoir of energy in the islet and augment its capacity to secrete hormones. The induction of CK-BB in the islet by other hormones could influence the secretion of insular hormones. Interorgan flux of the substrate creatine may be an undescribed mechanism of physiological regulation.

Epoxycreatine (N-(2,3-epoxypropyl)-N-amidinoglycine) is an affinity label of creatine kinase that irreversibly and completely inactivates the enzyme (Marletta, M. A., and Kenyon, G. L. (1979) J. Biol. Chem. 254, 1879-1886). To identify active site residues of rabbit muscle creatine kinase, the site of modification of it by epoxycreatine has been determined. Separation by high performance liquid chromatography of a tryptic digest of [14C]epoxycreatine-modified creatine kinase yielded two radiolabeled peptides. The larger of these consisted of amino acids Ala-266 through Arg-291 and was labeled with epoxycreatine at Cys-282. Attempts to purify completely the other labeled peptide were not successful; however, it was possible to obtain, by tandem mass spectrometry, a collision-induced dissociation spectrum of it from a mixture of several peptides. This peptide was a fragment (amino acids Val-279 through Arg-291) of the previously identified peptide and was also labeled at Cys-282. Model studies with cysteine and epoxycreatine have demonstrated that opening of the oxirane ring occurs by attack of the cysteine thiolate at the terminal carbon of the epoxide. These results are consistent with previous studies on the base lability of the label; however, a carboxyl group in the active site is not labeled, as had been previously suggested. These results provide evidence that Cys-282 is located in or near the creatine-binding site and will also be important in identifying and delineating the boundaries of the active site of creatine kinase.

Benzoylformate decarboxylase (BFDC) is a thiamin diphosphate- (ThDP-) dependent enzyme acting on aromatic substrates. In addition to its metabolic role in the mandelate pathway, BFDC shows broad substrate specificity coupled with tight stereo control in the carbon-carbon bond-forming reverse reaction, making it a useful biocatalyst for the production of chiral alpha-hydroxy ketones. The reaction of methyl benzoylphosphonate (MBP), an analogue of the natural substrate benzoylformate, with BFDC results in the formation of a stable analogue (C2alpha-phosphonomandelyl-ThDP) of the covalent ThDP-substrate adduct C2alpha-mandelyl-ThDP. Formation of the stable adduct is confirmed both by formation of a circular dichroism band characteristic of the 1',4'-iminopyrimidine tautomeric form of ThDP (commonly observed when ThDP forms tetrahedral complexes with its substrates) and by high-resolution mass spectrometry of the reaction mixture. In addition, the structure of BFDC with the MBP inhibitor was solved by X-ray crystallography to a spatial resolution of 1.37 A (PDB ID 3FSJ). The electron density clearly shows formation of a tetrahedral adduct between the C2 atom of ThDP and the carbonyl carbon atom of the MBP. This adduct resembles the intermediate from the penultimate step of the carboligation reaction between benzaldehyde and acetaldehyde. The combination of real-time kinetic information via stopped-flow circular dichroism with steady-state data from equilibrium circular dichroism measurements and X-ray crystallography reveals details of the first step of the reaction catalyzed by BFDC. The MBP-ThDP adduct on BFDC is compared to the recently solved structure of the same adduct on benzaldehyde lyase, another ThDP-dependent enzyme capable of catalyzing aldehyde condensation with high stereospecificity.

The crystal structure of mandelate racemase (MR) has been solved at 3.0-A resolution by multiple isomorphous replacement and subsequently refined against X-ray diffraction data to 2.5-A resolution by use of both molecular dynamics refinement (XPLOR) and restrained least-squares refinement (PROLSQ). The current crystallographic R-factor for this structure is 18.3%. MR is composed of two major structural domains and a third, smaller, C-terminal domain. The N-terminal domain has an alpha + beta topology consisting of a three-stranded antiparallel beta-sheet followed by an antiparallel four alpha-helix bundle. The central domain is a singly wound parallel alpha/beta-barrel composed of eight central strands of beta-sheet and seven alpha-helices. The C-terminal domain consists of an irregular L-shaped loop with several short sections of antiparallel beta-sheet and two short alpha-helices. This C-terminal domain partially covers the junction between the major domains and occupies a region of the central domain that is filled by an eight alpha-helix in all other known parallel alpha/beta-barrels except for the barrel domain in muconate lactonizing enzyme (MLE) [Goldman, A., Ollis, D. L., & Steitz, T. A. (1987) J. Mol. Biol. 194, 143] whose overall polypeptide fold and amino acid sequence are strikingly similar to those of MR [Neidhart, D. J., Kenyon, G. L., Gerlt, J. A., & Petsko, G. A. (1990) Nature 347, 692]. In addition, the crystal structure reveals that, like MLE, MR is tightly packed as an octamer of identical subunits. The active site of MR is located between the two major domains, at the C-terminal ends of the beta-strands in the alpha/beta-barrel domain. The catalytically essential divalent metal ion is ligated by three side-chain carboxyl groups contributed by residues of the central beta-sheet. A model of a productive substrate complex of MR has been constructed on the basis of difference Fourier analysis at 3.5-A resolution of a complex between MR and (R,S)-p-iodomandelate, permitting identification of residues that may participate in substrate binding and catalysis. The ionizable groups of both Lys 166 and His 297 are positioned to interact with the chiral center of substrate, suggesting that both of these residues may function as acid/base catalysts.(ABSTRACT TRUNCATED AT 400 WORDS)

The gene encoding rabbit muscle creatine kinase (CK) has been subcloned into a single plasmid, and the protein expressed in a soluble and functional form in Escherichia coli. The amino terminus, specific activity, and electrophoretic mobility of the E. coli-expressed creatine kinase are all identical with that of creatine kinase purified from rabbit skeletal muscle. Surprisingly, isoelectric focusing shows that the expressed protein displays no less heterogeneity than the tissue-purified material. The identification of the source(s) of this heterogeneity is important for the preparation of highly homogeneous material needed for structural studies and clinical applications. This issue also has implications for studies of the developmental regulation and tissue localization of the various CK genes. Our results allow us to eliminate some of the proposals, such as the presence of multiple alleles, alternative ribosomal initiation sites, and post-translational glycosylation or phosphorylation that have been suggested to explain the presence of the numerous isoforms present in apparently pure preparations of CK.

Benzoylformate decarboxylase from Pseudomonas putida ( Pp BFDC) is a thiamin diphosphate-dependent enzyme that carries out the nonoxidative decarboxylation of aromatic 2-keto acids. The x-ray structure of Pp BFDC suggested that Ser-26, His-70, and His-281 would play important roles in its catalytic mechanism, and the S26A, H70A, and H281A variants all exhibited greatly impaired catalytic activity. Based on stopped-flow studies with the alanine mutants, it was proposed that the histidine residues acted as acid-base catalysts, whereas Ser-26 was involved in substrate binding and played a significant, albeit less well defined, role in catalysis. While developing a saturation mutagenesis protocol to examine residues involved in Pp BFDC substrate specificity, we tested the procedure on His-281. To our surprise, we found that His-281, which is thought to be necessary for protonation of the carbanion/enamine intermediate, could be replaced by phenyl alanine with only a 5-fold decrease in k cat . Even more surprising were our subsequent observations ( i ) that His-70 could be replaced by threonine or leucine with approximately a 30-fold decrease in k cat / K m compared with a 4,000-fold decrease for the H70A variant and ( ii ) that Ser-26, which forms hydrogen bonds with the substrate carboxylate, could be replaced by threonine, leucine, or methionine without significant loss of activity. These results call into question the assigned roles for Ser-26, His-70, and His-281. Further, they demonstrate the danger in assigning catalytic function based solely on results with alanine mutants and show that saturation mutagenesis is a valuable tool in assessing the role and relative importance of putative catalytic residues.

The genes that encode the five known enzymes of the mandelate pathway of Pseudomonas putida (ATCC 12633), mandelate racemase (mdlA), (S)-mandelate dehydrogenase (mdlB), benzoylformate decarboxylase (mdlC), NAD(+)-dependent benzaldehyde dehydrogenase (mdlD), and NADP(+)-dependent benzaldehyde dehydrogenase (mdlE), have been cloned. The genes for (S)-mandelate dehydrogenase and benzoylformate decarboxylase have been sequenced; these genes and that for mandelate racemase [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540] are organized in an operon (mdlCBA). Mandelate racemase has regions of sequence similarity to muconate lactonizing enzymes I and II from P. putida. (S)-Mandelate dehydrogenase is predicted to be 393 amino acids in length and to have a molecular weight of 43,352; it has regions of sequence similarity to glycolate oxidase from spinach and ferricytochrome b2 lactate dehydrogenase from yeast. Benzoylformate decarboxylase is predicted to be 499 amino acids in length and to have a molecular weight of 53,621; it has regions of sequence similarity to enzymes that decarboxylate pyruvate with thiamin pyrophosphate as cofactor. These observations support the hypothesis that the mandelate pathway evolved by recruitment of enzymes from preexisting metabolic pathways. The gene for benzoylformate decarboxylase has been expressed in Escherichia coli with the trc promoter, and homogeneous enzyme has been isolated from induced cells.