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Human aspartoacylase is cloned and expressed in Pichia pastoris and the properties of this highly purified and fully active enzyme are examined. The chelation studies to remove the zinc have resulted in a reversible loss of catalytic activity, thereby establishing aspartoacylase as a zinc metalloenzyme.

The channeling of the substrate beta-aspartyl phosphate between aspartokinase and aspartate semialdehyde dehydrogenase in Escherichia coli is descrbed. Data suggest that aspartate semialdehyde dehydrogenase may facilitate the channeling of the intermediates between the nonconsecutive reactions of the enzyme.

Research examines the nonconsecutive reaction catalysis of the bifunctional aspartokinase-homoserine dehydrogenase isoenzymes from Escherichia coli as well as synthesized hybrid enzymes. Results show that in the hybrids, the kinase activity is sensitive to lysine and dehydrogenase is regulated by both threonine and lysine. The kinase domain is stabler than dehydrogenase domain.

Research has been conducted on the F65A/Y131C murine alpha-carbonic anhydrase V. The crystal structure of this anhydrase has been investigated in examining the catalytic proton shuttle features and the results are reported.

Research has been conducted on the aspartate-beta-semialdehyde dehydrogenase. The structure of this dehydrogenase from Escherichia coli has been investigated via crystallography in the presence of its coenzyme and substrate analogue and the results are reported.

Researchers have investigated potential active site residues for L-aspartate ammonia-lyase from Escherichia coli on the basis of their proximity to a cleft which contains the functionally significant lysine 327. It was possible to use site-directed mutagenesis to test the five most likely residues in the assumed active site cleft. Arg 29 was assigned a substrate binding role, and it was concluded that the beta-carboxyl group of the substrate was the most likely site of interaction.

Researchers have succeeded in identifying the structure of aspartase from Escherichia coli to 2.8 angstrom. Sequence alignments of the fumarase superfamily and mutagenesis studies have made it possible to locate the likely active site of aspartase in a region which contains side chains from three different subunits. The high substrate specificity of aspartase indicates that the enzyme must bind both the alpha- and beta-carboxylate groups.

The substrate specificity of homoserine kinase was analyzed by utilizing a series of structural analogs of L-homoserine exhibiting site-directed mutagenesis in specific functional groups. Sequence homology studies of the structural analogs of L-homoserine during homoserine kinase-catalyzed reactions indicated the role of arginine-234 in the binding of the carboxyl group. Furthermore, the beta- and gamma-carbons of the hydroxyl group were phosphorylated by homoserine kinase which can also accommodate large hydrophobic groups at the carboxyl end of the substrate.

A study, to identify residues that play an important role in the catalytic activity of aspartate-beta-semialdehyde dehydrogenase (ASA DH), by site -directed mutagenesis, compares ASA DH structure with D-glyceraldehyde-3-phosphate dehydrogenases (GAPDH) to select correct targets for mutagenesis. ASA DH catalyzes the oxidative phosphorylation of an aldehyde substrate and has an essential cysteine residue that is involved in enzymatic reaction.

Site-directed mutagenic study of the L-aspartase enzyme of Escherichia coli suggests that the cysteine-273 residue is modified by the treatment of L-aspartase with L-aspartate-beta-semialdehyde. Thus, cysteine-273 serves as an active-site nucleophile that enables attack of an activated double bond in the enzyme-bound product involved in catalysis in L-aspartase.

Substrate analogue L-aspartate beta-semialdehyde (L-ASA) is a mechanism-based inactivator of L-aspartase from Escherichia coli that catalyzes the deamination of L-ASA to give fumaric acid semialdehyde (FAA) and NH(sub 4)(super +). L-ASA acts as a mechanism-based inactivator by binding at the active site of the enzyme. FAA binds more cohesively to the enzyme-activator-metal ion complex than to the free enzyme form. This confirms the coordination in binding between the activator site and the active site of L-aspartase.

The kinetics of formation and isomerization of phosphato complexes of uridine 5'-di and triphosphates to platinum(II) were reported. The resulting products were characterized with high performance liquid chromatogrphy, circular dichroism and two-dimensional nuclear magnetic resonance spectroscopy. The results show that the isomerization proceeds through a rate-limiting ring-opening. When the chelate ring is opened, deligation and phosphate hydrolysis can also occur, resulting in free UDP, UMP and orthophosphate bound to platinum.

The substrate specificity of aspartokinase I from Escherichia coli was examined by kinetic, isotope labeling and nuclear magnetic resonance studies. It was shown that the alpha-amino group is an essential binding group. In addition, beta-derivatized structural analogues, such as beta-amide, beta-hydroxamate andbeta-esters, were found to be alternative substrates. These beta-derivatized analogues are capable of binding to aspartokinase through a reversal of regiospecificity. The result raises the possibility of using these alternative substrates to synthesize antimetabolites for the enzymes that are downstream inthe aspartate biosynthetic pathway.

The specificity of the anion binding site of aspartate-beta-semialdehyde dehydrogenase was investigated. The objective was to identify alternative substrates that can substitute for phosphate and competitive inhibitors with the ability to stabilize the acyl enzyme intermediate to allow structural characterization. Several oxyanions interact with L-aspartate-beta-semialdehyde dehydrogenase either as an alternative substrate or as a competitive inhibitor.

The structural studies of human aspartoacylase (hACY2) brain enzyme complexed with a tetrahedral intermediate analogue, N-phosphonomethyl-L-aspartate bound in the active site is reported. The structural assignments, combined with kinetic characterization of several active site mutants, provide new insights into the catalytic mechanism of aspartoacylase and understand the defects in the clinical mutants that lead to Canavan disease.

The structure and stability of the binuclear manganese cluster is examined. It was found that for catalytic activity, the stability of the manganese cluster is important to catalyze the hydrolysis of L-arginine to form urea.