Acid−Base Chemical Mechanism of Homocitrate Synthase from Saccharomyces cerevisiae

Abstract

Homocitrate synthase (acetyl-coenzyme A:2-ketoglutarate C-transferase; E.C. 2.3.3.14) catalyzes the condensation of AcCoA and alpha-ketoglutarate to give homocitrate and CoA. The enzyme was found to be a Zn-containing metalloenzyme using inductively coupled plasma mass spectrometry. Dead-end analogues of alpha-ketoglutarate were used to obtain information on the topography of the alpha-ketoglutarate binding site. The alpha-carboxylate and alpha-oxo groups of alpha-ketoglutarate are required for optimum binding to coordinate to the active site Zn. Optimum positioning of the alpha-carboxylate, alpha-oxo, and gamma-carboxylate of alpha-ketoglutarate is likely mimicked by the location in space of the 2-carboxylate, pyridine nitrogen, and 4 carboxylate of pyridine 2,4-dicarboxylate. The pH dependence of the kinetic parameters was determined to obtain information on the chemical mechanism of homocitrate synthase. The V profile is bell shaped with slopes of 1 and -1, giving pKa values of 6.7 and 8.0, while V/K(AcCoA) exhibits a slope of 2 on the acidic side with an average pKa value of 6.6 and a slope of -2 on basic side of the profile with an average pKa value of 8.2. The V/K(alpha-Kg) pH-rate profile exhibits a single pKa of 6.9 on the acidic side and two on the basic side with an average value of 7.8. The pH dependence of the Ki for glyoxylate, a competitive inhibitor vs alpha-ketoglutarate, gives a pKa of 7.1 for a group, required to be protonated for optimum binding. Data suggest a chemical mechanism for the enzyme in which alpha-ketoglutarate first binds to the active site Zn via its alpha-carboxylate and alpha-oxo groups, followed by acetyl-CoA. A general base then accepts a proton from the methyl of acetyl-CoA, and a general acid protonates the carbonyl of alpha-ketoglutarate in the formation of homocitryl-CoA. The general acid then acts as a base in deprotonating Zn-OH2 in the hydrolysis of homocitryl-CoA to give homocitrate and CoA. A solvent deuterium kinetic isotope effect of 1 is measured for homocitrate synthase, while a small pH-independent primary kinetic deuterium isotope effect (approximately 1.3) is observed using deuterioacetyl-CoA. Data suggest rate-limiting condensation to form the alkoxide of homocitryl-CoA, followed by hydrolysis to give products.

Evaluation of Lysine Biosynthesis as an Antifungal Drug Target: Biochemical Characterization of Aspergillus fumigatus Homocitrate Synthase and Virulence Studies

“…Interestingly, the EDTA sensitivity of the P. chrysogenum enzyme was much less pronounced, and the enzyme showed only 30% inhibition at 5 mM EDTA (26). Although S. cerevisiae homocitrate synthase is also Mg independent, it has been assumed to be a Zn-containing metalloenzyme, and the Zn 2ϩ might be involved directly in catalysis (46). Since A. fumigatus HcsA was 25% inhibited in the presence of 1 M ZnSO 4 , a Zn 2ϩ requirement of A. fumigatus HcsA appears unlikely.…”

Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

“…2B). The N-terminal domain (residues 1-300) is composed of a canonical (␣/␤) 8 TIM barrel (residues 35-300) with additional secondary structural elements flanking and inserted within the barrel. In the SpHCS⅐2-OG complexes, an N-terminal extension (residues 1-34) consisting of a short ␤-strand (␤0) followed by a 3 10 helix (3 10 1) precedes the ␤1 strand of the TIM barrel in the SpHCS⅐2-OG complexes, whereas the residues preceding the 3 10 1 helix are disordered in the apoenzyme structure.…”

“…In the SpHCS⅐2-OG complexes, an N-terminal extension (residues 1-34) consisting of a short ␤-strand (␤0) followed by a 3 10 helix (3 10 1) precedes the ␤1 strand of the TIM barrel in the SpHCS⅐2-OG complexes, whereas the residues preceding the 3 10 1 helix are disordered in the apoenzyme structure. Within the TIM barrel, several short 3 10 helices are interspersed throughout the (␣/␤) 8 fold, and a region of mixed topology is inserted between ␤4 and ␣4. In the SpHCS⅐2-OG complexes, this region is fully ordered in the electron density maps and consists of the 3 10 4 helix followed by a ␤-hairpin motif formed by ␤4.1-␤4.2, whereas these structural elements are disordered in the apoenzyme structure (see below).…”

“…Catalytic Mechanism-The catalytic mechanism of HCS is believed to proceed via a mixed aldol Claisen condensation reaction (8) (Fig. 6A).…”

“…The kinetic mechanism of Saccharomyces cerevisiae HCS (ScHCS) Lys20 has been reported to obey an ordered Bi-Bi model in which 2-OG binding precedes AcCoA followed by the sequential release of the products CoA and homocitrate (7). The proposed reaction mechanism of HCS proceeds via a mixed aldol Claisen condensation that involves enzyme acid-and base-catalyzed steps (8), which is the same mechanism used by the citric acid cycle enzyme citrate synthase (CS) (9). Insights into this mechanism have been derived from analyses of the crystal structure of Mycobacterium tuberculosis ␣-isopropylmalate synthase (␣-IPMS) encoded by the LeuA gene.…”