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The penultimate reaction in the oxidative degradation of nicotinate (vitamin B(3)) to fumarate in several species of aerobic bacteria is the hydrolytic deamination of maleamate to maleate, catalyzed by maleamate amidohydrolase (NicF). Although it has been considered a model system for bacterial degradation of N-heterocyclic compounds, only recently have gene clusters that encode the enzymes of this catabolic pathway been identified to allow detailed investigations concerning the structural basis of their mechanisms. Here, the Bb1774 gene from Bordetella bronchiseptica RB50, putatively annotated as nicF, has been cloned, and the recombinant enzyme, overexpressed and purified from Escherichia coli, is shown to catalyze efficiently the hydrolysis of maleamate to maleate and ammonium ion. Steady-state kinetic analysis of the reaction by isothermal titration calorimetry (ITC) established k(cat) and K(M) values (pH 7.5 and 25 °C) of 11.7 ± 0.2 s(-1) and 128 ± 6 μM, respectively. The observed K(D) of the NicF·maleate (E·P) complex, also measured by ITC, is approximated to be 3.8 ± 0.4 mM. The crystal structure of NicF, determined at 2.4 Å using molecular replacement, shows that the enzyme belongs to the cysteine hydrolase superfamily. The structure provides insight concerning the roles of potential catalytically important residues, most notably a conserved catalytic triad (Asp29, Lys117, and Cys150) observed in the proximity of a conserved non-proline cis-peptide bond within a small cavity that is likely the active site. On the basis of this structural information, the hydrolysis of maleamate is proposed to proceed by a nucleophilic addition-elimination sequence involving the thiolate side chain of Cys150.

The electron distribution within the coenzyme or coenzyme-substrate conjugate needs to be properly regulated during the catalytic process of aspartate aminotransferase (AspAT). Asn194 and Tyr225 may function in regulating the electron distribution through hydrogen-bonding to O(3') of the coenzyme, pyridoxal 5'-phosphate (PLP) or pyridoxamine 5'-phosphate (PMP). The roles of Tyr225 have already been explored by site-directed mutagenesis (Inoue et al., 1991; Goldberg et al., 1991). In the present studies, the mutant enzymes Asn194-->Ala and Asn194-->Ala + Tyr225-->Phe were analyzed kinetically and spectroscopically and were compared with the wild-type and Tyr225-->Phe enzymes. The kinetic studies showed that Asn194 is not essential for AspAT catalysis, although the Kd values for the substrates were increased by 10- to 50-fold upon the replacement of Asn194. The measurements of the absorption and fluorescence excitation spectra revealed that the ratio of an enolimine to a ketoenamine form was considerably increased as a tautomeric form of the protonated PLP in the active site of the double mutant enzyme. The pH-pKd relationship for the binding of maleate to AspAT could be explained by a simple thermodynamic cycle where only one ionizing group (the imine nitrogen of the internal aldimine bond) affects the binding of maleate. The analyses of the pH-pKd curves for the wild-type and mutant enzymes showed that (i) the hydrogen bond between O(3') of PLP and Asn194 is weakened by the binding of maleate to AspAT, while the hydrogen bond between O(3') and Tyr225 is not changed, and that (ii) the replacement of Asn194 causes some effect hampering the binding of maleate.(ABSTRACT TRUNCATED AT 250 WORDS)

The aspartate aminotransferase mutant Y70F exhibits kcat = 8% and kcat/KM = 2% of the wild type values for the transamination of aspartate and alpha-ketoglutarate. The affinity of the enzyme for the noncovalently bound inhibitor maleate is reduced 17-fold by the mutation, while only a 2.5-fold reduction is observed for alpha-methylaspartate, which forms a stable, covalent external aldimine. The high population of the quinonoid intermediate formed in the reaction of the wild type with beta-hydroxyaspartate is more than 75% diminished by the mutation. The values of the Y70F C alpha-H kinetic isotope effects for the aspartate reaction are larger than those of wild type (DV = 2.4 vs 1.52; D(V/K) = 2.5 vs 1.7). Conversely, the Y70F value of D(V/K) for the glutamate reaction is decreased compared to wild type (1.75 vs 2.5). These results, combined with previous studies of Lys258 mutants, eliminate Tyr70 as an essential component of the catalytic apparatus, with the caveat that the functionally of the deleted hydroxyl group is possibly replaced by a water molecule.

Maleylacetone cis-trans-isomerase isolated from Vibrio 01 binds glutathione strongly; Km = 1.4 X 10(-4) M. Oxidized glutathione and S-methylglutathione are competitive inhibitors, KI = 9.4 X 10(-4) and 1.2 X 10(-3) M, respectively. Based on these interactions, three different glutathione-bound agarose affinity adsorbents were synthesized and tested. Affinity chromatography of the isomerase with one of these affords 70- to 100-fold purifications. In separate syntheses, portions of the affinity arm were prepared and examined as to their inhibitory properties in the enzyme-catalyzed reaction. The fragment, containing glutathione bound through its sulfur to the carbon chain, is a powerful competitive inhibitor for glutathione (KI = 6 X 10(-5) M). The results described suggest that the isomerase binds glutathione through the backbone of the tripeptide and that the thiol group is required for activity. The initial velocity patterns of the enzyme-catalyzed reaction resulting from simultaneous variation of glutathione and maleylacetone concentrations were examined in the absence and presence of inhibitors resembling glutathione. The observed kinetic patterns suggest an ordered sequence of binding maleylacetone first followed by glutathione.