Identification of the bromopyruvate-sensitive glutamate within the active site of 2-keto-3-deoxygluconate-6-P aldolase

Bromopyruvate inactivates 2-keto-3-deoxygluconate-6-P aldolase by a mechanism in which the reagent is incorporated by esterification. A tryptic peptide derived from inactivated enzyme has the sequence Thr-Leu-Glu¹-Val-Thr-Leu-Arg. Derivatization of the γ-carboxyl of the single glutamate by bromopyruvate was confirmed by Lossen rearrangement in which the glutamate γ-ester was converted to 2,4-diamino butyrate.     

On the interaction of 2-keto-3-deoxygalactonate-6P with 2-keto-3-deoxygluconate-6P aldolase ofPseudomonas putida. Evidence for enzyme-mediated,noncatalytic C-C bond turnover

2-Keto-3-deoxygluconate-6P (KDPG) aldolase ofPseudomonas putida mediates the cleavage of, as well as the condensation of, pyruvate andd-glyceraldehyde-3P (GaP) yielding, 2-keto-3-deoxygalactonate-6P (KDPGal) as side reactions of normal catalysis. These are visualized at high levels of aldolase. KDPGal cleavage occurs with aV _max that is 1/5000 that for KDPG cleavage. TheKm for KDPGal is 0.2 mM, with aK _i of 0.85 mM. The E-KDPGal complex is reductively inactivated having aKd of 0.55 mM. TheV/K value for KDPG cleavage is 2.0×10⁸ sec^?1, while the value for KDPGal cleavage is 1220 sec^?1. The difference in first-order rate constants of 164,000-fold argues that a step in the cleavage of KDPGal mediated by the enzyme is uncatalyzed. The enzyme is reductively inactivated by trapping the E-pyruvate, E-KDPG, or E-KDPGal complex. The enzyme can also be inactivated by reductive trapping of a catalytically nonproductive E-glyceraldehyde-3P complex. This latter occurs with aKd for GaP of 20 mM and a rate constant equivalent to a limiting half-time of 1110 sec at 1 mM cyanoborohydride. Reductive inactivation half-times in the presence of high GaP/KDPG ratios were the sum of both E-GaP and E-KDPG trapping by cyanoborohydride so that the inactivation rate due to KDPG could be determined. It was found at 1 mM cyanoborohydride that the limiting half-time for the E-KDPG complex was 2382 sec. The corresponding value for the E-KDPGal complex was 215 sec. Consequently, the E-KDPGal complex is 11 times more sensitive to reductive derivativation than is the E-KDPG complex. This is interpreted to show that the enzyme binds the KDPGal in a “normal” step forming a ketimine. However, turnover to the eneamine with resultant C-C bond cleavage is uncatalyzed. For the case of KDPGal synthesized by KDPG aldolase, it is argued that the pyruvate eneamine is bound to the active site, which can be attacked by GaP with its aldehyde carbon in the catalytically nonproductive conformation as a side reaction, presumably forming a tertiary complex. Spontaneous protonation of the resultant alcoholate anion would generate KDPGal. The data are interpreted to support an argument that catalytic proton turnover at the OH of C-4 of KDPG is required for normal catalysis, and that this step, which catalytically interconverts ketimine/eneamine, imposes steric constraints controlling the overall stereochemistry of the reaction.     

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.     

On the interaction of tritiated 2-ketobutyrate with 2-keto-3-deoxygluconate-6P aldolase ofPseudomonas putida. Evidence suggesting enzyme-mediated,uncatalyzed tritium exchange

2-Keto-3-deoxygluconate-6P aldolase ofPseudomonas putida mediates exchange between hydrogen isotope at the methylene carbon of 2-ketobutyrate and water. This occurs with aK _m of 20 mM, 100 times the corresponding value for pyruvate, and a V_max approximating 1/710 that of KDPG cleavage. Ketobutyrate is competitive with both pyruvate and 2-keto-3-deoxygluconate-6P for the enzyme. In addition, there is no evidence for C-C synthesis between ketobutyrate andd-glyceraldehyde-3P. A comparison of relativeV/K values for hydrogen exchange shows pyruvate to be 17,600 times better as a substrate than ketobutyrate. The detritiation of [3-³H]ketobutyrate is stereochemically random. In addition, the reaction proceeds with ak _H/k _T isotope effect of 15.3, consistent with C-H bond turnover being rate-determining. The E-ketobutyrate complex is reductively trapped, inactivating the enzyme. Reductive inactivation kinetics of E-ketobutyrate compared to E-pyruvate suggests more of the complex may be partitioned to ketimine in the ketobutyrate case than in the pyruvate case. A mechanism is considered in which ketobutyrate is bound as a ketimine in an orientation such that the active site acid/basic group cannot mediate catalytic ketimine/eneamine interconversion. Thus, exchange would result from hydrogen ionization at C-3′ of the ketimine, a slow spontaneous step compared to overall complex turnover. This noncatalyzed deprotonation would explain dissymmetry in exchange, the poorV/K compared to pyruvate, and a large tritium isotope effect.     

The conformation of catalytically functional Schiff's bases: the pyruvate--lysine ketimine of 2-keto-3-deoxygluconate-6-phosphate aldolase of Pseudomonas putida

The reduction stereochemistry of the Schiff's base formed between pyruvate and the ?-amino of the catalytic lysine of 2-keto-3-deoxygluconate-6-P-phosphate aldolase of Pseudomonas putida was investigated. Reduction was stereoselective yielding 55.73% N⁶-[(1R)-and 44.27% N⁶-[(1S)-1-carboxyethyl]-S-lysine. Thus the reducing agent predominated slightly at the si face of the ketimine carbon. For comparison, the reduction stereochemistry of the pyruvate-lysine ketimine formed on d-amino acid oxidase during d-alanine turnover at pH 8.5 was also investigated. In this case reduction was random, consistent with nonactive site participation in that transimination reaction generating the ketimine, as postulated by other investigators of this enzyme.     

The interaction of bromopyruvate with the active site of 2-keto-3-deoxygluconate-6-phosphate aldolase ofPseudomonas saccharophila: Kinetics and stereochemistry

The interaction of bromopyruvate with the active site of 2-keto-3-deoxygluconate-6-P aldolase ofPseudomonas saccharophila was investigated. The reagent inactivates the enzyme, exhibiting saturation kinetics and competition with pyruvate. The minimal inactivation half-time was 6 min, equivalent to a first-order rate constant of 0.115 min^?1. The concentration of bromopyruvate giving the half-maximal inactivation rateK _inact was 50 mM. TheK _s value of pyruvate as a competitive inhibitor was 0.85 mM. The enzyme asymmetrically detritiates (3RS)-[3? ³H ₂ ]bromopyruvate, forming, in water, (3S)-[3-³H,H]bromopyruvate. This stereochemistry is also exhibited by 2-keto-6-deoxygalactonate-6-P aldolase isolated from the same organism as well as the 2-keto-3-deoxygluconate-5-P aldolase ofP. putida. Over a range of [3-³H]bromopyruvate concentrations affecting the inactivation rate, the ratio of nanomoles reagent catalytically turned over per unit of enzyme inactivated remained constant at 14:1, providing evidence that both catalysis and alkylation occur at the same protein site.     

Formation and cleavage of 2-keto-3-deoxygluconate by 2-keto-3-deoxygluconate aldolase of Aspergillus niger.

2-Keto-3-deoxygluconate aldolase of Aspergillus niger, an enzyme that has not been reported previously, was purified 468-fold. Maximal activity was obtained at pH 8.0 and 50 C. The enzyme exhibited relative stereochemical specificity with respect to glyceraldehyde. The Km values for 2-keto-3-deoxygluconate, glyceraldehyde, and pyruvate were 10, 13.3, and 3.0 mM, respectively. The effects of some compounds and inhibitors on enzyme activity were examined. Stability of the enzyme under different conditions was investigated. The equilibrium constant was about 0.33 X 10(-3) M.     

Structure of 2-keto-3-deoxy-6-phosphogluconate aldolase at 2.8 Å resolution

The structure of 2-keto-3-deoxy-6-phosphogluconate aldolase has been extended to 2.8 Å resolution from 3.5 Å resolution by multiple isomorphous replacement methods using three heavy-atom derivatives and anomalous Bijvoet differences to 6 Å resolution (〈m〉 = 0.72). The replacement phases were improved and refined by electron density modification procedures coupled with inverse transform phase angle calculations. A Kendrew model of the molecule was built, which contained all 225 residues of a recently determined amino acid sequence, whereas only 173 were accounted for at 3.5 Å resolution. The missing residues were found to be part of the interior of the molecule and not simply an appendage. The molecule folds to form an eight-strand α/β-barrel structure strikingly similar to triosephosphate isomerase, the A-domain of pyruvate kinase and Taka amylase. With a knowledge of the sequence, the nature of the interfaces of the two kinds of crystallographic trimers have been examined, from which it was concluded that the choice of trimers selected in the 3.5 Å resolution work was probably correct for trimers in solution. The active site region has been established from the position of the Schiff base forming Lys144 but it has not been possible to confirm it conclusively in independent derivative experiments. An apparent anomaly exists in the location of Glu56 (about 25 Å from Lys144). The latter has been reported to assist in catalysis.     

Slow-binding inhibition of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase is a key enzyme in the Entner-Doudoroff pathway of bacteria. It catalyzes the reversible production of KDPG from pyruvate and D-glyceraldehyde 3-phosphate through a class I Schiff base mechanism. On the basis of aldolase mechanistic pathway, various pyruvate analogues bearing beta-diketo structures were designed and synthesized as potential inhibitors. Their capacity to inhibit aldolase catalyzed reaction by forming stabilized iminium ion or conjugated enamine were investigated by enzymatic kinetics and UV-vis difference spectroscopy. Depending of the substituent R (methyl or aromatic ring), a competitive or a slow-binding inhibition takes place. These results were examined on the basis of the three-dimensional structure of the enzyme.     

The singly-wound parallel beta barrel: a proposed structure for 2-keto-3-deoxy-6-phosphogluconate aldolase.

Two short local reconnections in the backbone chain tracing of 2-keto-3-deoxy-6-phosphogluconate aldolase suffice to make it an 8-stranded parallel β barrel whose size, shape, topology, and connection handedness match those of triose phosphate isomerase and of the first domain of pyruvate kinase. It is proposed that this singly-wound parallel β barrel is in fact the tertiary structure of the aldolase subunit.     

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.     

Map location of 2-keto-3-deoxy-6-P-gluconate aldolase negative mutations in E. coli K12

Summary We have isolated mutants unable to utilize any of the following carbon sources: D-glucuronate, D-galacturonate, and D-gluconate. During the metabolism of each of these substrates, these mutants accumulate 2-keto-3-deoxy-6-P-gluconate; they were subsequently found to be deficient for the enzyme: 2-keto-3-deoxy-6-P-gluconate aldolase. This deficiency results in an inhibition of growth on various carbonsources when a hexuronate or gluconate is present in the medium.By means of conjugation experiments, we have mapped the locus (eda) of these negative mutations between his and old D. A cotransduction was found with old D (16%) and with edd (96%); the order of loci appears to be edd-eda-old D. The relationship of this locus eda with a structural gene for aldolase is suggested.

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