Showing papers by "Christopher T. Walsh published in 1981"

Mechanistic studies on the pyridoxal phosphate enzyme 1-aminocyclopropane-1-carboxylate deaminase from Pseudomonas sp

Abstract: The enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACPC deaminase) from a pseudomonad is a pyridoxal phosphate (PLP) linked catalyst which fragments the cyclopropane substrate to alpha-ketobutyrate and ammonia [Honma, M., & Shimomura, T. (1978) Agric. Biol. Chem. 42, 1825]. Enzymatic incubations in D2O yield alpha-ketobutyrate with one deuterium at the C-4 methyl group and one deuterium at one of the C-3 prochiral methylene hydrogens. Stereochemical analysis of the location of the C-3 deuteron was accomplished by in situ enzymatic reduction to (2S)-2-hydroxybutyrate with L-lactate dehydrogenase and conversion to the phenacyl ester. The C-3 hydrogens of the (2S)-2-hydroxybutyryl moiety are fully resolved in a 250-MHz NMR spectrum. Absolute assignment of 3S and 3R loci was obtained with phenacyl (2S,3S)-2-hydroxy[3-2H]butyrate generated enzymatically by D-serine dehydratase action on D-threonine. ACPC deaminase shows a stereoselective outcome with a 3R:3S deuterated product ratio of 72:28. 2-Vinyl-ACPC is also a fragmentation substrate with exclusive regiospecific cleavage to yield the straight-chain keto acid product 2-keto-5-hexenoate. The D isomer of vinylglycine is processed to alpha-ketobutyrate and ammonia at 8% the Vmax of ACPC, while L-vinylglycine is not a substrate. It is likely that ACPC and D-vinylglycine yield a common intermediate--the vinylglycine-PLP-p-quinoid adduct--which is then protonated sequentially at C-4 and then C-3 to account for the observed deuterium incorporation. The D isomers of beta-substituted alanines (fluoroalanine, chloroalanine, and O-acetyl-D-serine) partition between catalytic elimination and enzyme inactivation. Each shows a different partition ratio, arguing against the common aminoacrylyl-PLP as the inactivating species.

Characteristics of .beta.,.beta.-difluoroalanine and .beta.,.beta.,.beta.-trifluoroalanine as suicide substrates for E. coli B alanine racemase

Abstract: The alanine racemase from Escherichia coli B has been shown to process DL isomers of beta -fluoroalanine as suicide substrates with an identical partitioning ratio for each enantiomer of 820 catalytic eliminations of HF per enzymatic inactivation event [Wang, E., & Walsh, C. T. (1978) Biochemistry 17, 1313], suggesting the aminoacrylate--PLP complex as a common, symmetrical partitioning species. In an attempt to vary the partition ratio, an index of killing efficiency, systematically the beta, beta-difluoroalanine and beta, beta, beta-trifluoroalanine isomers have now been evaluated for substrate processing, suicidal inactivation kinetics and partitioning ratio, and stability of inactive, derivatized enzyme forms. Both difluoroalanine isomers show high Km values (116 mM for D, 102 mM for L) in catalytic HF loss to form fluoropyruvate. The Vmax for the D isomer is about 14-fold higher than that for the L isomer. Limiting inactivation rate constants, calculated from kcat and observed partition ratios of 5000 and 2600, respectively, are 2.2 min-1 for D-difluoroalanine and 0.33 min-1 for L-difluoroalanine. For comparison, DL-trifluoroalanine turns over less than 10 times per enzyme molecule inactivated and so is a very efficient suicide substrate. The estimated inactivation rate constant is less than or equal to 1.0 min-1. These data are analyzed in terms of partitioning behavior of the monofluoro- and difluoroaminoacrylate--PLP complexes as partitioning intermediates for turnover or for racemase inactivation. While mono- and trifluoroalanines yield stable inactive species, the difluoroalanine isomers produce labile enzyme derivatives, and regain of catalytic activity is analyzed in terms of the anticipated oxidation state at the beta carbon of the substrate fragment adducted to the enzyme.

Mechanistic studies on reactions of bacterial methionine gamma-lyase with olefinic amino acids.

Abstract: Methionine gamma-lyase (EC 4.4.1.11), which catalyzes the formation of methanethiol, alpha-ketobutyrate, and ammonia from L-methionine (eq 1), promotes the oxidative deamination of several four- and five-carbon olefinic amino acids (1-5). With the exception of vinylglycine (1), the Vmax rates of keto acid formation from the unsaturated substrate analogues are substantially lower than that for processing of methionine to alpha-ketobutyrate; vinylglycine is deaminated to ketobutyrate and ammonia with a Vmax twice that for L-methionine turnover. L-Allylglycine, L-2-amino-3-trans-pentenoate, and L-2-amino-3-cis-pentenoate (2, 4, 5) are all converted to 2-keto-pentanoic acid (alpha-ketovalerate). L-2-Amino-3-cis-pentenoate (5) is also a time-dependent, irreversible inactivator of the enzyme. None of the other substrate analogues tested appears to inactivate the enzyme. Spectral analysis of the enzymatic reaction with cis isomer 5 reveals the formation of a high-wavelength chromophore (lambda max = 550 nm ) which implies that a beta, gamma-unsaturated pyridoxal p-quinoid (VI) accumulates. No such absorbing species appears to form during the reaction of trans isomer 4 with methionine gamma-lyase. But a 550-nm chromophore develops when both 4 and 5 are reacted with Al(NO3)3 and pyridoxal methochloride in methanolic KOH. It would appear that the geometry of the protein and the olefinic amino acid as an intermediate enzyme-substrate adduct controls the kinetics of reaction, such that azaallylic isomerization becomes selectively rate determining for reaction with 5. When this isomerization is slow, an accumulating Michael-type acceptor (VI) could lead to the observed irreversible inactivation of the enzyme.

Reconstitution of Escherichia coli membrane vesicles with D-amino acid dehydrogenase.

Abstract: When purified D-amino acid dehydrogenase [Olsiewski, P. J., Kaczorowski, G. J., & Walsh, C. T. (1980) J. Biol. Chem. 255, 4487] is incubated with right-side-out membrane vesicles from Escherichia coli, the enzyme binds to the membrane in a time- and concentration-dependent manner. As a result, the vesicles acquire the ability to oxidize D-alanine and catalyze D-alanine-dependent active transport. Similarly, incubation of D-amino acid dehydrogenase with inside-out vesicles results in binding of enzyme and D-alanine oxidase activity. Antibody inhibition studies indicate that the enzyme is bound exclusively to the inner cytoplasmic surface of the membrane in native vesicles (i.e., membrane vesicles prepared from cells induced for D-amino acid dehydrogenase). In contrast, similar studies with reconstituted vesicles demonstrate that enzyme binds to the surface exposed to the medium regardless of the orientation of the membrane. Thus, enzyme bound to right-side-out vesicles is located on the opposite side of the membrane from where it is normally found. Remarkably, in the presence of D-alanine, reconstituted right-side-out and inside-out vesicles generate electrochemical proton gradients of similar magnitude but opposite polarity, indicating that enzyme bound to either surface of the membrane is physiologically functional. The results suggest that vectorial proton translocation via the respiratory chain occurs at a point distal to the site where electrons enter the respiratory chain from the primary dehydrogenase, a conclusion that is inconsistent with the notion that the dehydrogenase forms part of a proton-translocating loop.