Proton transfer in methylmalonyl-CoA epimerase from Propionibacterium shermanii. Studies with specifically tritiated (2R)-methylmalonyl-CoA as substrate.

(2R)-Methyl[2-3H]malonyl-CoA was used as the substrate for methylmalonyl-CoA epimerase from Propionibacterium shermanii, under conditions where the (2S)-methylmalonyl-CoA product was removed enzymically as fast as it was formed, and the fate of the label was monitored at different extents of reaction. Very little, if any, tritium is found attached to the C-2 position in the (2S)-epimer product (isolated as propionyl-CoA). Evidently, the hydrogen atom of the new C-H bond in the product is essentially solvent-derived. The rate of tritium release into the solvent is lower than the rate of product formation, and shows a primary kinetic tritium-isotope effect on kcat./Km of 2.3 +/- 0.1. The specific radioactivity of the remaining substrate rises slowly during the epimerase-catalysed reaction, and this provides an independent estimate of the primary kinetic tritium-isotope effect on kcat./Km of 1.6 +/- 0.5. These results, taken together, indicate that the mechanistic pathway of the epimerase-catalysed reaction resembles that established for proline racemase [Cardinale & Abeles, (1968) Biochemistry 7, 3970-3978], in which two enzyme bases are involved in catalysis. One base removes the proton from the substrate, the second provides the new proton, and there is no fast isotopic exchange between enzyme-bound intermediates and solvent protons.     

Crystal structure of substrate complexes of methylmalonyl-CoA mutase.

X-ray crystal structures of methylmalonyl-CoA mutase in complexes with substrate methylmalonyl-CoA and inhibitors 2-carboxypropyl-CoA and 3-carboxypropyl-CoA (substrate and product analogues) show that the enzyme-substrate interactions change little during the course of the rearrangement reaction, in contrast to the large conformational change on substrate binding. The substrate complex shows a 5'-deoxyadenine molecule in the active site, bound weakly and not attached to the cobalt atom of coenzyme B12, rotated and shifted from its position in the substrate-free adenosylcobalamin complex. The position of Tyralpha89 close to the substrate explains the stereochemical selectivity of the enzyme for (2R)-methylmalonyl-CoA.     

Triosephosphate isomerase: energetics of the reaction catalyzed by the yeast enzyme expressed in Escherichia coli

Triosephosphate isomerase from bakers' yeast, expressed in Escherichia coli strain DF502(p12), has been purified to homogeneity. The kinetics of the reaction in each direction have been determined at pH 7.5 and 30 degrees C. Deuterium substitution at the C-2 position of substrate (R)-glyceraldehyde phosphate and at the 1-pro-R position of substrate dihydroxyacetone phosphate results in kinetic isotope effects on kcat of 1.6 and 3.4, respectively. The extent of transfer of tritium from [1(R)-3H]dihydroxyacetone phosphate to product (R)-glyceraldehyde phosphate during the catalyzed reaction is only 3% after 66% conversion to product, indicating that the enzymic base that mediates proton transfer is in rapid exchange with solvent protons. When the isomerase-catalyzed reaction is run in tritiated water in each direction, radioactivity is incorporated both into the remaining substrate and into the product. In the "exchange-conversion" experiment with dihydroxyacetone phosphate as substrate, the specific radioactivity of remaining dihydroxyacetone phosphate rises as a function of the extent of reaction with a slope of about 0.3, while the specific radioactivity of the products is 54% that of the solvent. In the reverse direction with (R)-glyceraldehyde phosphate as substrate, the specific radioactivity of the product formed is only 11% that of the solvent, while the radioactivity incorporated into the remaining substrate (R)-glyceraldehyde phosphate also rises as a function of the extent of reaction with a slope of 0.3. These results have been analyzed according to the protocol described earlier to yield the free energy profile of the reaction catalyzed by the yeast isomerase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereospecificity of malonyl-CoA decarboxylase, acetyl-CoA carboxylase, and fatty acid synthetase from the uropygial gland of goose

Malonyl-CoA decarboxylase from the uropygial gland of goose decarboxylated (R,S)-methylmalonyl-CoA at a slow rate and introduced 3H from [3H]2O into the resulting propionyl-CoA. Carboxylation of this labeled propionyl-CoA by propionyl-CoA carboxylase from pig heart and acetyl-CoA carboxylase from the uropygial gland completely removed 3H. Repeated treatment of (R,S)-[methyl-14C]methylmalonyl-CoA with the decarboxylase converted 50% of the substrate into propionyl-CoA, whereas (S)-methylmalonyl-CoA, generated by both carboxylases, was completely decarboxylated. Radioactive (R)- (S), and (R,S)-methylmalonyl-CoA were equally incorporated into fatty acids by fatty acid synthetase from the uropygial gland. The residual methylmalonyl-CoA remaining after fatty acid synthetase reaction on (R,S)-methylmalonyl-CoA was also racemic. These results show that: (a) the decarboxylase is stereospecific, (b) replacement of the carboxyl group by hydrogen occurs with retention of configuration, (c) acetyl-CoA carboxylase of the uropygial gland generates (S)-methylmalonyl-CoA from propionyl-CoA, and (d) fatty acid synthetase is not stereospecific for methylmalonyl-CoA.     

Gamma-butyrobetaine hydroxylase: stereochemical course of the hydroxylation reaction

The stereochemical course of the aliphatic hydroxylation of gamma-butyrobetaine by calf liver and by Pseudomonas sp AK1 gamma-butyrobetaine hydroxylases has been determined. With [3(RS)-3-3H]-gamma-butyrobetaine or [3(R)-3-3H]-gamma-butyrobetaine as substrate, a rapid and significant loss of tritium to the medium occurred. On the other hand, with [3(S)-3-3H]-gamma-butyrobetaine, only a negligible release of tritium to the aqueous medium was observed. Indeed, on hydroxylation of [3(S)-3-2H]-gamma-butyrobetaine by either the calf liver or bacterial hydroxylase, the isolated product L-carnitine was found to have retained all of the deuterium initially present in the 3(S) position. Since the absolute configuration of the product L-carnitine has been determined to be R, such results are only compatible with a hydroxylation reaction that proceeded with retention of configuration. With [methyl-14C,3(R)-3-3H]-gamma-butyrobetaine as substrate for the calf liver hydroxylase, the percentage of tritium retained in the [methyl-14C]-L-carnitine product was determined as a function of percent reaction. The results of these studies indicated that pro-R hydrogen atom abstraction exceeded 99.9%. Experiments using racemic [methyl-14C,3(RS)-3-3H]-gamma-butyrobetaine as substrate yielded similar results and additionally allowed us to estimate alpha-secondary tritium kinetic isotope effects of 1.10 and 1.31 for the bacterial and calf liver enzymes, respectively. These results are discussed within the context of the radical mechanism for gamma-butyrobetaine hydroxylase previously proposed [Blanchard, J. S., & Englard, S. (1983) Biochemistry 22, 5922], and the required topographical arrangement of enzymic oxidant and substrate is illustrated.     

Thymidylate synthetase. Catalysis of dehalogenation of 5-bromo- and 5-iodo-2'-deoxyuridylate.

Tymidylate synthetase catalyzes the facile dehalogenation of 5-bromo-2'-deoxyuridylate (BrdUMP) and 5-iodo-2'-deoxyuridylate )IdUMP) to give 2'-deoxyuridylate (dUMP), the natural substrate of the enzyme. The reaction does not require folate cofactors and stoichiometrically consumes 2 equiv of thiol. In addition to dUMP, a minor product is formed during the debromination of BrdUMP which has been identified as a 5-alkylthio derivative formed by displacement of bromide ion by thiolate. The reaction has been found to proceed with a substantial alpha-secondary inverse tritium isotope effect (kT/kH = 1.212--1.258) with [2-14C,6-3H]-BrdUMP as the substrate. Similarly, an inverse tritiumisotope effect of 1.18 was observed in the nonenzymatic chemical counterpart of this reaction, the cysteine-promoted dehalogenation of [2-14C,6-3H]-5-bromo-2'-deoxyuridine. Previous evidence for the mechanism of action of this enzyme has rested largely on chemical model studies and on information obtained from its stoichiometric interaction with the inhibitor 5-fluoro-2'-deoxyuridylate. The magnitude of the secondary isotope effect during the enzymatic dehalogenation described here provides direct proof for nucleophilic catalysis and formation of 5,6-dihydroprimidine intermediates in a reaction catalyzed by thymidylate synthetase.     

Stereochemistry of the methylmalonyl-CoA decarboxylation reaction

The steric course of the decarboxylation of (S)-methylmalonyl-CoA to propionyl-CoA, catalyzed by the biotin-dependent sodium pump methylmalonyl-CoA decarboxylase of Veillonella alcalescens was determined. The decarboxylation of (S)-methylmalonyl-CoA in 3H2O yielded (R)-[2-3H]propionyl-CoA; and the decarboxylation of (S)-[2-3H]methylmalonyl-CoA in H2O produced (S)-[2-3H]propionyl-CoA. The results demonstrate retention of configuration during the decarboxylation reaction. The substrate stereochemistry of methylmalonyl-CoA decarboxylase is thus the same as that of all other biotin-containing enzymes investigated.     

Stereospecific abstraction of epsilon-pro-R-hydrogen of L-lysine by L-lysine epsilon-dehydrogenase from Agrobacterium tumefaciens

The stereochemical aspects of the L-lysine epsilon-dehydrogenase reaction were examined with (6R)-L-[6-3H]lysine and (6S)-DL-[6-3H]lysine. When (6S)-DL-[6-3H]lysine was used as a substrate, the tritium was found in the product, delta 1-piperideine-6-carboxylate. In contrast, the radioactivity from (6R)-L-[6-3H]lysine was not retained in the product. Thus, the pro-R hydrogen at the prochiral C-6 carbon of L-lysine is specifically abstracted by the enzyme: the enzyme behaves stereochemically as an amino acid D-dehydrogenase.     

On the mechanism of action of methylmalonyl-CoA mutase. Change of the steric course on isotope substitution

When (methyl-2H3)methylmalonyl-CoA was reacted with partially purified methylmalonyl-CoA mutase, 1H-NMR revealed that about 24% of the migrating deuterium was lost after 88% conversion. When [methyl-3H]methylmalonyl-CoA was incubated with highly purified methylmalonyl-CoA mutase, tritium exchange with the medium depended on added methylmalonyl-CoA epimerase. With highly purified preparations of methylmalonyl-CoA mutase, effective tritium exchange from [5'-3H]adenosylcobalamin to water required the addition of methylmalonyl-CoA epimerase and of substrate (e.g. succinyl-CoA). By addition of [14C]succinyl-CoA to a partially purified preparation of methylmalonyl-CoA mutase, it was shown that the mutase binds one substrate molecule very tightly. Coupling the mutase reaction with the transcarboxylase reaction and using variously labelled succinyl-CoA as substrate, revealed that only (2R)- and not (2S)-methylmalonyl-CoA will be formed by the mutase with a kinetic isotope effect of 3.5 using (2H4)succinyl-CoA. When (1-13C) propionyl-CoA was reacted with a mixture of highly purified methylmalonyl-CoA carboxylase, epimerase and mutase, 13C-NMR signals were obtained for the thioester carbonyl of succinyl-CoA (relative intensity 100%) and of methylmalonyl-CoA (5%) as well as for the carboxyl of free succinic acid (27%) and of succinyl-CoA (less than 4.5%). Thus very little, if any, migration of the CoA from one carboxyl to the other appears to take place. (1,4-13C2)Succinic acid and (1,4-13C2)succinyl-CoA were synthesised and their 13C-NMR chemical shifts were exactly determined. Evidence is provided for a strict stereospecificity of the mutase toward the (2R)-epimer of methylmalonyl-CoA and for an incomplete stereospecificity toward the two diastereotopic 3-H atoms of succinyl-CoA. The latter, combined with a high intramolecular isotope discrimination, causes rapid washing-out of the migrating 2H and 3H to water and slow washing-in from the medium. Whenever migration of protium from the sterically less preferred 3-pro(S)- position of succinyl-CoA occurs and simultaneously a heavy isotope is maneuvered from the migratable 3-pro(R)- position into the labile alpha-position of methylmalonyl-CoA, the substitution by the COSCoA group takes place with inversion of configuration. When the sterically preferred 3-pro(R)-hydrogen atom migrates, the previously reported stereochemical retention occurs. A mechanistic and stereochemical scheme is discussed that fully accounts for all observations.     

Changes in the free energy profile of glutamate mutase imparted by the mutation of an active site arginine residue to lysine

Arginine 100 plays an important role in substrate recognition in adenosylcobalamin-dependent glutamate mutase. We have examined how the mutation of this residue to lysine affects the partitioning of tritium, incorporated at the exchangeable position of the coenzyme, between substrate and product. We find that partitioning of tritium back to the substrate predominates in the mutant enzyme, regardless of whether the reaction is run in the forward or reverse direction. This contrasts with the behavior of the wild-type enzyme in which tritium partitions equally between substrate and product, independent of the direction of the reaction. From this we conclude that the mutation significantly impairs the ability of the enzyme to catalyze the rearrangement of substrate radical to product radical. The results illustrate the importance of electrostatic interactions in stabilizing free radical intermediates in this class of enzymes.     

Crystal structure of methylmalonyl-coenzyme A epimerase from P. shermanii: a novel enzymatic function on an ancient metal binding scaffold

BACKGROUND: Methylmalonyl-CoA epimerase (MMCE) is an essential enzyme in the breakdown of odd-numbered fatty acids and of the amino acids valine, isoleucine, and methionine. Present in many bacteria and in animals, it catalyzes the conversion of (2R)-methylmalonyl-CoA to (2S)-methylmalonyl-CoA, the substrate for the B12-dependent enzyme, methylmalonyl-CoA mutase. Defects in this pathway can result in severe acidosis and cause damage to the central nervous system in humans. RESULTS: The crystal structure of MMCE from Propionibacterium shermanii has been determined at 2.0 A resolution. The MMCE monomer is folded into two tandem betaalphabetabetabeta modules that pack edge-to-edge to generate an 8-stranded beta sheet. Two monomers then pack back-to-back to create a tightly associated dimer. In each monomer, the beta sheet curves around to create a deep cleft, in the floor of which His12, Gln65, His91, and Glu141 provide a binding site for a divalent metal ion, as shown by the binding of Co2+. Modeling 2-methylmalonate into the active site identifies two glutamate residues as the likely essential bases for the epimerization reaction. CONCLUSIONS: The betaalphabetabetabeta modules of MMCE correspond with those found in several other proteins, including bleomycin resistance protein, glyoxalase I, and a family of extradiol dioxygenases. Differences in connectivity are consistent with the evolution of these very different proteins from a common precursor by mechanisms of gene duplication and domain swapping. The metal binding residues also align precisely, and striking structural similarities between MMCE and glyoxalase I suggest common mechanisms in their respective epimerization and isomerization reactions.     

Reaction mechanism of glutamate racemase, a pyridoxal phosphate-independent amino acid racemase.

Glutamate racemase of Pediococcus pentosaceus contained no cofactor, and was completely inactivated by a thiol reagent. The role of a cysteine residue in the enzyme reaction was studied by chemical modification. The modification of this cysteine residue resulted in a concomitant loss of activity. DL-Glutamate protected the enzyme from inactivation. The inactivated enzyme was reactivated by addition of dithiothreitol. The racemization in 2H2O showed an overshoot in the optical rotation of glutamate before the substrate was completely racemized. This indicates that the removal of alpha-hydrogen is the rate determining step. During the racemization of D- or L-glutamate in 3H2O, tritium was incorporated preferentially into the product. Glutamate is racemized by the enzyme probably through a two base mechanism.     

Mechanism of action of glutaryl-CoA and butyryl-CoA dehydrogenases. Purification of glutaryl-CoA dehydrogenase

Glutaryl-CoA dehydrogenase, a flavoprotein, catalyzes the reaction -OOCCH3CH2--CH2COSR (FAD leads to FADH2) leads to CH3CH = CHCOSR + CO2 (SR = CoA or pantetheine). With the isolated enzyme, a dye serves as the final electron acceptor. The enzyme from Pseudomonas fluorescens (ATCC 11250) has been purified to homogeneity. It was established with appropriate isotopic substitutions that the proton which is added to the gamma position of the product, subsequent to decarboxylation, is not derived from the solvent but is derived from the alpha position of the substrate. Under conditions where no net conversion of substrate occurs, i.e., in the absence of electron acceptor, the enzyme catalyzes the exchange of the beta hydrogen of the substrate with solvent protons. Butyryl-CoA dehydrogenase (M. elsedenii), which catalyzes an analogous reaction, catalyzes the exchange of both the alpha and beta hydrogens with solvent protons in the absence of electron acceptor. Glutaryl-CoA dehydrogenase and butyryl-CoA dehydrogenase are irreversibly inactivated by the substrate analogues 3-butynoylpantetheine and 3-pentynoylpantetheine. These inactivators do not form an adduct with the flavin and probably react with a nucleophile at the active site. Upon inactivation, the spectrum of the enzyme-bound flavin is essentially unchanged, and the flavin can be reduced by Na2S2O4. We suggest that inactivation involves intermediate allene formation. We proposed that these results support an oxidation mechanism for glutaryl-CoA dehydrogenase and butyryl-CoA dehydrogenase which is initiated by proton abstraction. With glutaryl-CoA dehydrogenase, the base, which abstracts the substrate alpha proton, is shielded from the solvent and is then used to protonate the carbanion (CH2--CH--CHCOSCoA) formed after oxidation and decarboxylation.     

Triosephosphate isomerase: removal of a putatively electrophilic histidine residue results in a subtle change in catalytic mechanism

An important active-site residue in the glycolytic enzyme triosephosphate isomerase is His-95, which appears to act as an electrophilic component in catalyzing the enolization of the substrates. With the techniques of site-directed mutagenesis, His-95 has been replaced by Gln in the isomerase from Saccharomyces cerevisiae. The mutant isomerase has been expressed in Escherichia coli strain DF502 and purified to homogeneity. The specific catalytic activity of the mutant enzyme is less than that of wild type by a factor of nearly 400. The mutant enzyme can be resolved from the wild-type isomerase on nondenaturing isoelectric focusing gels, and an isomerase activity stain shows that the observed catalytic activity indeed derives from the mutant protein. The inhibition constants for arsenate and for glycerol phosphate with the mutant enzyme are similar to those with the wild-type isomerase, but the substrate analogues 2-phosphoglycolate and phosphoglycolohydroxamate bind 8- and 35-fold, respectively, more weakly to the mutant isomerase. The mutant enzyme shows the same stereospecificity of proton transfer as the wild type. Tritium exchange experiments similar to those used to define the free energy profile for the wild-type yeast isomerase, together with a new method of analysis involving 14C and 3H doubly labeled substrates, have been used to investigate the energetics of the mutant enzyme catalyzed reaction. When the enzymatic reaction is conducted in tritiated solvent, the mutant isomerase does not catalyze any appreciable exchange between protons of the remaining substrate and those of the solvent either in the forward reaction direction (using dihydroxyacetone phosphate as substrate) or in the reverse direction (using glyceraldehyde phosphate as substrate). However, the specific radioactivity of the product glyceraldehyde phosphate formed in the forward reaction is 31% that of the solvent, while that of the product dihydroxyacetone phosphate formed in the reverse reaction is 24% that of the solvent. The deuterium kinetic isotope effects observed with the mutant isomerase using [1(R)-2H]dihydroxyacetone phosphate and [2-2H]glyceraldehyde 3-phosphate are 2.15 +/- 0.04 and 2.4 +/- 0.1, respectively. These results lead to the conclusion that substitution of Gln for His-95 so impairs the ability of the enzyme to stabilize the reaction intermediate that there is a change in the pathways of proton transfer mediated by the mutant enzyme. The data allow us more closely to define the role of His-95 in the reaction catalyzed by the wild-type enzyme, while forcing us to be alert to subtle changes in mechanistic pathways when mutant enzymes are generated.     

Ab initio models for receptor-ligand interactions in proteins. 4. Model assembly study of the catalytic mechanism of triosephosphate isomerase

The catalytic mechanism of triosephosphate isomerase (TIM) was investigated with ab initio quantum mechanical calculations. Electrostatic interactions between the quantum mechanical active site and the protein and solvent environment were modeled using the finite difference Poission-Boltzman method. The complexes of TIM with the substrate dihydroxyacetone phosphate (DHAP), five possible intermediates and the product glyceraldehyde-3-phosphate (GAP) were optimized in the active-site model at the 3-21G^(*) level and energy profile for the proton abstraction from DHAP by the active-site Glu167 was calculated at the MP2/3-21G^(*)//3-21G^(*) level. Calculated energetics of the enzyme reaction were found to be in reasonable agreement with the experimental findings. Calculations revealed that an enediol of the substrate is a probable intermediate in the enzyme reaction. It was suggested that the proton abstracted from the substrate by the active-site glutamate goes to the carbonyl oxygen of the substrate producing enediol intermediate either directly or after it is exchanged with solvent. © 1996 Wiley-Liss, Inc.     

Tritium partitioning and isotope effects in adenosylcobalamin-dependent glutamate mutase.

Tritiated adenosylcobalamin, labeled at the exchangeable position, has been used to investigate the partitioning of tritium between substrate and product in the reaction catalyzed by glutamate mutase. The isotope partitions between glutamate and methylaspartate in nearly 1:1 ratio, regardless of the direction in which the overall reaction is proceeding. This is consistent with a free-energy profile in which the interconversion of the intermediate glutamyl and methylaspartyl radicals is rapid relative to the transfer of tritium from 5'-deoxyadenosine to either substrate or product. Initial velocity measurements have been used to measure the tritium isotope effects for the transfer of tritium from adenosylcobalamin to product in each direction. The isotope effect is 21 for the formation of glutamate and 19 for the formation of methylasparate. The large magnitude of these isotope effects makes it likely that the rate-determining step may be altered by the substitution of tritium for hydrogen in the reaction. The results of these experiments are compared with previous isotope effect measurements made on other adenosylcobalamin-dependent enzymes.     

Rate-limiting steps and role of active site Lys443 in the mechanism of carbapenam synthetase

Carbapenam synthetase (hereafter named CPS) catalyzes the formation of the beta-lactam ring in the biosynthetic pathway to (5R)-carbapen-2-em-3-carboxylate, the simplest of the carbapenem antibiotics. Kinetic studies showed remarkable tolerance to substrate stereochemistry in the turnover rate but did not distinguish between chemistry and a nonchemical step such as product release or conformational change as being rate-determining. Also, X-ray structural studies and modest sequence homology to beta-lactam synthetase, an enzyme that catalyzes the formation of a monocyclic beta-lactam ring in a similar ATP/Mg2+-dependent reaction, implicate K443 as an essential residue for substrate binding and intermediate stabilization. In these experiments, we use pH-rate profiles, deuterium solvent isotope effects, and solvent viscosity measurements to examine the rate-limiting step in this complex overall process of substrate adenylation and intramolecular ring formation. Mutagenesis and chemical rescue demonstrate that K443 is the general acid visible in the pH-rate profile of the wild-type CPS-catalyzed reaction. On the basis of these results, we propose a mechanism in which the rate-limiting step is beta-lactam ring formation coupled to a protein conformational change and underscore the role of K443 throughout the reaction.     

Properties of aspartate racemase, a pyridoxal 5'-phosphate-independent amino acid racemase.

Aspartate racemase from Streptococcus thermophilus contains no pyridoxal 5'-phosphate or other cofactors such as FAD, NAD+, and metal ions. It was affected by neither carbonyl reagents such as hydroxylamine nor sodium borohydride but was strongly inhibited by iodoacetamide and other thiol reagents. Aspartate, cysteate, and cysteine sulfinate were the only substrates. The Km values for L- and D-aspartate were 35 and 8.7 mM, respectively. The enzyme catalyzed the exchange of alpha-hydrogen of the substrate with the solvent hydrogen. Racemization of L-aspartate in 2H2O showed an overshooting in the optical rotation of aspartate before the substrate was fully racemized. This shows that the removal of alpha-hydrogen of the substrate is at least partially rate-determining. When L- or D-aspartate was incubated with aspartate racemase in tritiated water, tritium was incorporated preferentially into the product enantiomer. The results strongly suggest that aspartate racemase contains two hydrogen acceptors.     

Alpha-secondary tritium kinetic isotope effects indicate hydrogen tunneling and coupled motion occur in the oxidation of L-malate by NAD-malic enzyme

The NAD-malic enzyme from Ascaris suum catalyzes the divalent metal ion-dependent oxidative decarboxylation of L-malate to give pyruvate and CO2, with NAD+ as the oxidant. Alpha-secondary tritium kinetic isotope effects were measured with NAD+ or APAD+ and L-malate-2-H(D) and several different divalent metal ions. The alpha-secondary tritium kinetic isotope effects are slightly higher than 1 with NAD+ and L-malate as substrates, much larger than the expected inverse isotope effect for a hybridization change from sp2 to sp3. The alpha-secondary tritium kinetic isotope effects are reduced to values near 1 with L-malate-2-D as the substrate, regardless of the metal ion that is used. Data suggest the presence of quantum mechanical tunneling and coupled motion in the malic enzyme reaction when NAD+ and malate are used as substrates. Isotope effects were also measured using the D/T method with NAD+ and Mn2+ as the substrate pair. A Swain-Schaad exponent of 2.2 (less than the value of 3.26 expected for strictly semiclassical behavior) is estimated, suggesting the presence of other slow steps along the reaction pathway. With APAD+ and Mn2+ as the substrate pair, inverse alpha-secondary tritium kinetic isotope effects are observed, and a Swain-Schaad exponent of 3.3 is estimated, consistent with rate-limiting hydride transfer and no quantum mechanical tunneling or coupled motion. Data are discussed in terms of the malic enzyme mechanism and the theory developed by Huskey for D/T isotope effects as an indicator of tunneling [Huskey, W. P. (1991) J. Phys. Org. Chem. 4, 361-366].     

New substrates for the radioassay of angiotensin converting enzyme of endothelial cells in culture.

To develop means of measuring angiotensin converting enzyme of endothelial cells in culture, we have synthesized benzoyl-Phe-Ala-Pro-OH (I), benzoyl-Pro-Phe-Arg-OH (II) and benzoyl-Gly-His-Leu-OH (III), each bearing a 3H-atom on the para-position of its benzoyl moiety. All three of the acylated tripeptides are substrates for the enzyme. Substrate I exhibits the lowest Km (12.5 micrometer) and yields the most sensitive assay: the enzyme of 10(6) cells can be measured in a 30 min incubation at 37 degrees C. Radiolabelled reaction product is separated from substrate by extraction of acidified reaction mixture with an organic solvent, and the rate of formation of product can be quantified by liquid scintillation counting of the organic phase. Substrate III can also be used to measure angiotensin converting enzyme of cells but requires longer incubations (180--240 min) and high salt concentrations (0.75 M Na2SO4). Substrate II is not specific: it is hydrolyzed by more than one enzyme of endothelial cells.