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H P Meloche

Publications and source records attributed to H P Meloche.

At least 19 recordsLinked to original sources
PubMed1979

Interaction of the chiral pyruvate analog, 2-keto-3-bromobutyrate, with pyruvate lyases. 2-Keto-3-deoxygluconate-6-phosphate aldolase of Pseudomonas putida.

The enzyme 2-keto-3-deoxygluconate-6-P aldolase of Pseudomonas putida is inactivated by one of the chiral forms of 2-keto-(3RS)-3-bromobutyric acid (bromoketobutyrate). The inactivation shows saturation kinetics and competition with pyruvate. The minimal inactivation half-time is 4 min and that concentration of bromoketobutyrate half-saturating the enzyme is 2 mM. (3RS)-[3-3H]bromoketobutyrate is catalytically detritiated during enzyme inactivation. A kinetic analysis of rates gave data consistent with both catalysis and inactivation occurring at a single protein site, the catalytic site. The enzyme only detritiates one of the two optical isomers of bromoketobutyrate, and that form which is detritiated also alkylates the catalytic site. The inactive isomer of reagent degrades, with inversion, to L-lactate so that the chiral form specific for the enzyme is 2-keto-(3S)-3-bromobutyrate. Thus, as is the case with bromopyruvate, the enzyme catalyzes protonation of the re face at C-3 of the enzyme-reagent eneamine. As a result, bromoketobutyrate could serve as a chiral probe for stereochemical constraints of selected pyruvate-specific lyase active sites.

Aldehyde-Lyases↗
PubMed1977

Specificity of 2-keto-3-deoxygluconate-6-P aldolase for open chain form of 2-keto-3-deoxygluconate-6-P.

2-Keto-3-deoxygluconate-6-P exists as an euqilibrium of three forms at 25 degrees measurable by 13C NMR: alpha-furanose anomer (41%), beta-furanose anomer (50%), and open chain keto (9%). The three forms are interconverted rapidly (greater than 0.5 s-1) so that the unidirectional rates of furanose ring opening and closing can be quantitated by an NMR line broadening method. The 2-keto-3-deoxygluconate aldolase is specific for only one of these forms, the open chain keto form. The rates for ring opening calculated from the rapid kinetic enzyme system compare closely with those obtained with the NMR method.

Aldehyde-Lyases↗
PubMed1977

Magnitude of the equilibrium isotope effect on carbon-tritium bond synthesis.

The exchange of tritium from 3HOH into the methyl group of pyruvate catalyzed by 6-phospho-2-keto-3-deoxygluconate aldolase (6-phospho-2-keto-3-deoxy-D-gluconate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.14) of Pseudomonas putida shows an equilibrium isotope effect of 0.78. From this value and the deuterium effect on the fumarase equilibrium (Thomson, J.F. (1960) Arch. Biochem. Biophys. 90, 1), one can calculate by use of the relative fractionation factors of Hartshorn and Shiner (Hartshorn, S.R. and Shiner, Jr., V.J. (1972) J. Am. Chem. Soc. 94, 9002), fractionation factors for transfer of deuterium or tritium from water to a number of organic molecules of interest.

Aldehyde-Lyases↗
PubMed1975

The stereochemistry at carbon 3 of pyruvate lyase condensation products. 2-Keto-3-deoxygluconate-6-phosphate aldolase.

In a condensation between [3-3H3]pyruvate and D-glyceraldehyde-3-P as catalyzed by 2-keto-3-deoxygluconate-6-P aldolase (EC 4.1.2.14) of Pseudomonas putida, C--C synthesis occurred appreciably faster than C--3H bond breaking. Since tritium is present in tritiated pyruvate in tracer amounts, this result showed hydrogen isotope discrimination in pyruvate deprotonation and suggests enolpyruvate generation to be at least partially rate-limiting in the condensation reaction. Consequently, in a condensation reaction between [3-3H, 2H,H]pyruvate of known chirality and D-glyceraldehyde-3-P, the newly synthesized C--C bond would be enriched for at what was the C--H bond of chiral pyruvate, discriminating against the C--2H and C--3H bonds. Additional studies showed that condensations between (3S)-[3-3H, 2H,H]- or (3R)-[3-3H, 2H,H]pyruvate and D-glyceraldehyde-3-P yielded predominantly (3S)- or (3R)-2-keto-3-deoxy[3-3H, 2H]gluconate-6-P, respectively. By comparison with sterochemical models, it was concluded that condensation occurred with retention of configuration at C-3. Thus in the turnover of substrates as catalyzed by this enzyme, both the exchanging proton from water and D-glyceraldehyde-3-P attack the same face of the enzyme-bound pyruvyleneamine.

Aldehyde-Lyases↗
PubMed1975

The sterochemistry at carbon 3 of pyruvate lyase condensation products. 2-Keto-3-deoxygluconate 6-phosphate and 2-keto-3-deoxygalactonate-6-phosphate aldolase of Pseudomonas saccharophila.

In Pseudomonas saccharophila 2-keto-3-deoxygalactonate-6-P aldolase (EC 4.1.2.21) is induced by growth on galatose while 2-keto-3-deoxygluconate-6-P aldolase (EC 4.1.2.14) is constitutive. These enzymes catalyze identical reactions except for the configuration fixed at C-4 during the condensation reaction. It was found with each enzyme that in a condensation between [3-3H3]pyruvate and D-glyceraldehyde-3-P, the respective condensation products were formed 8 to 10 times faster than tritium was released to water. Since pyruvate deprotonation is obligatory for condensation, the above result requires a hydrogen isotope effect in enolpyruvate formation, which must be then at least partially rate limiting for C--C synthesis. Further, condensation between D-glyceraldehyde-3-P and (3R)-[3-3H, 2H,H]pyruvate or (3S)-[3-3H, 2H,H]pyruvate, as catalyzed by each enzyme, enriched for (3R)- and (3S)-3-3H, 2H-labeled condensation product, respectively. Thus, each enzyme catalyzes C--C and C--H synthesis with retention of configuration at C-3. This shows that the active sites of both enzymes are asymmetric since solutes can only approach a single face of the bound pyruvyl enolate. In addition, the respective aldehyde specific portions of the two active sites must have opposite chiralities, with respect to each other, for correctly orienting the carbonyl faces of the incoming D-glyceraldehyde-3-P, to generate the correct configuration at C-4 of the respective condensation products.

Aldehyde-Lyases↗
PubMed1975

Bromopyruvate inactivation of 2-keto-3-deoxy-6-phosphogalactonate aldolase of Pseudomonas saccharophila. Kinetics and stereochemistry.

The enzyme 2-keto-3-deoxy-6-phosphogalactonate aldolase of Pseudomonas saccharophila is inactivated by the substrate analog beta-bromopyruvate, which satisfies several criteria of being an active site directed reagent. The inactivation exhibits saturation kinetics, and both bromopyruvate and pyruvate (substrate) compete for free enzyme. Upon prolonged incubation, inactivation is virtually complete. The Kinact for bromopyruvate is 12 mM and the minimum inactivation half-time is 16 min with a k of 0.0433 min minus 1. Bromopyruvate is also a substrate for the enzyme in that 3(R,S)-[3-3H2]bromopyruvate is asymmetrically detritiated by the enzyme yielding 3(S)-[3-3H,H]bromopyruvate concomitant with inactivation. At various concentrations of bromopyruvate which affect the inactivation rate, the ratio of nanomoles of bromopyruvate turned over/unit of enzyme inactivated remains constant averaging 12:1, consistent with both inactivation and catalysis occurring at a single protein site, the catalytic site. The above value does not take into account a possible hydrogen isotope effect and is not thus an absolute value. The stereochemistry of bromopyruvate turnover catalyzed by this enzyme is the same as that for 2-keto-3-deoxy-6-phosphogluconate aldolase of P. putida. This fact provides the first evidence that the pyruvate-specific portions of the two active sites may have evolved from a common precursor.

Aldehyde-Lyases↗
PubMed1973

Aldolase catalysis: single base-mediated proton activation.

The enzyme, 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, catalyzes several reactions, the natural ones being (i) the exchange of hydrogen atoms of the methyl groups of pyruvate with protons of the solvent (C-H synthesis) and (ii) the reversible condensation of pyruvate with D-glyceraldehyde-3-phosphate (C-C synthesis). Previous work has provided chemical evidence for the occurrence of a protein-bound carboxylate group adjacent to the Schiff's base-forming lysine in the active site geometry. This carboxylate could provide the basic group postulated to participate in proton activation catalyzed by aldolases. With the use of three-dimensional models, it is shown that simple rotation about a carbon-carbon bond of the side chain will allow the base to assume the two positions necessary for proton activation in either the C-H synthesis or the C-C synthesis catalyzed by KDPG aldolase. This single base hypothesis provides a model wherein all reagents can approach a single face of the active site and is consistent with the stereochemistry thought to occur in the aldolase reaction.

Aldehyde-Lyases↗