Catalytic strategy of citrate synthase: effects of amino acid changes in the acetyl-CoA binding site on transition-state analog inhibitor complexes

Abstract

Acetyl-CoA enol has been proposed as an intermediate in the citrate synthase (CS) reaction with Asp375 acting as a base, removing a proton from the methyl carbon of acetyl-CoA, and His274 acting as an acid, donating a proton to the carbonyl [Karpusas, M., Branchaud, B., & Remington, S.J. (1990) Biochemistry 29, 2213]. CS-oxaloacetate (OAA) complexes with the transition-state analog inhibitor, carboxymethyl-CoA (CMCoA), mimic those with acetyl-CoA enol. Asp375 and His274 interact intimately with the carboxyl of the bound inhibitor. While enzymes in which these residues have been changed to other amino acids have very low catalytic activity, we find that they retain their ability to form complexes with substrates and the transition-state analog inhibitor. In comparison with the value of the chemical shift of the protonated CMCoA carboxyl in acidic aqueous solutions or its value in the wild-type ternary complex, the values in the Asp375 mutants are unusually low. Model studies suggest that these low values result from complete absence of one hydrogen bond partner for the Gly mutant and distortions in the active site hydrogen bond systems for the Glu mutant. The high affinity of Asp375Gly-OAA for CMCoA suggests that the unfavorable proton uptake required to stabilize the CMCoA-OAA ternary complex of the wild-type enzyme [Kurz, L.C., Shah, S., Crane, B.R., Donald, L.J., Duckworth, H.W., & Drysdale, G.R. (1992) Biochemistry (preceding paper in this issue)] is not required by this mutant; the needed proton is most likely provided by His274. This supports the proposed role of His274 as a general acid.(ABSTRACT TRUNCATED AT 250 WORDS)

The Partial Substrate Dethiaacetyl-Coenzyme A Mimics All Critical Carbon Acid Reactions in the Condensation Half-Reaction Catalyzed by Thermoplasma acidophilum Citrate Synthase

“…The maximal rate of complexation ( k max ) is comparable to the rate of acetyl-CoA- and citryl-CoA-dependent turnover by D317G– Tp CS, and somewhat faster than HDX (phosphate and D 2 O inhibition exaggerates the true difference in rates) 2. The low rate of acetyl-CoA dependent turnover in D317G– Tp CS is consistent with the widely accepted conclusion that Asp317 accepts the proton removed from the acetyl-CoA methyl group during the condensation reaction (3, 19, 24, 26, 29). The parallel suppression of the dethiaacetyl-CoA HDX rate in D317G– Tp CS demonstrates that Asp317, assisted by other parts of the catalytic apparatus, has a similar function during dethiaacetyl-CoA complexation.…”

The Ubiquitin Ligase SCFUcc1 Acts as a Metabolic Switch for the Glyoxylate Cycle

“…To examine these possibilities, we analyzed a mutant Cit2 that is defective in oxaloacetate binding and thus lacks citrate synthase activity. The histidine residue at 274 in PCS is located near the oxaloacetate/acetyl-CoA binding pocket ( Figure 6A, PCS, colored green) and functions as a proton donor during the condensation reaction (Kurz et al, 1992;Kurz et al, 1998). Replacement of this critical histidine with glycine (H274G mutant of PCS) causes a defect in oxaloacetate binding, an inefficient conformational change from open to closed form, and a loss of citrate synthase activity (Kurz et al, 1992(Kurz et al, , 1998; Evans et al,…”

Regulation of Hsp90 ATPase Activity by the Co-chaperone Cdc37p/p50

“…2 The K d values determined from differential CD data were in very good agreement with those obtained from calorimetry. A similar approach has been used to determine the K d values of binary complexes by a non-linear regression method (27), using the difference CD obtained by subtracting the CD of the total added concentration of both host and ligand from the observed CD of the host-ligand complex.…”