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Citrate synthase from the thermophile Thermoplasma acidophilum is less efficient than the same enzyme from mammals. The bacterial enzyme may be less flexible because of the required thermal stability of the enzyme or its substrate.

The effects of changes in three catalytic residues on the relative stabilities of some of the intermediates and transition states in the citrate synthase reaction are presented. Data indicate that the residues are involved in the stabilization of one or more of the transition states in the multistep citrate synthase reaction. However, none of these mutants can activate the carbonyl of oxaloacetate by polarization. All changes have effects on more than one step in the catalytic cycle. Surprisingly, the energetics of citrate synthase are tightly coupled.

Raman difference spectroscopy was used to determine the spectra of purine ribonucleoside and one of its analogues in both the free state and the adenosine deaminase-bound state in solution. The Raman bands were identified using ab initio methods. Results reveal that the enzyme catalyzes the hydration of the bound inhibitor leading to the disruption of a specific hydrogen bond as revealed by vibrational analyses.

The optimal induction conditions and solubility levels of each mutant porcine CS genes expressed in Escherichia coli were analyzed to determine the mechanisms of citrate synthase-catalyzed proton transfer. Citrate synthase activity was sensitive to steric factors, differences in the bond length of the C-S-C unit of the natural thioester substrate and the C-C-C of the ketone analog. Furthermore, citrate synthase-catalyzed proton transfer was also dependent on the activation of the oxaloacetate substrate through the polarization of the carbonyl bonds.

His238, a conserved amino acid situated in hydrogen-bonding distance from C-6 of the substrate in the active site of murine adenosine deaminase (mADA) and suspected of being involved in catalysis, was altered into an alanine, a glutamate, and an arginine using site-directed mutagenesis. The Ala and Glu substitutions failed to produce changes of the secondary or tertiary structure, while the Arg mutation resulted in local perturbations in tertiary structure and quenched the emission of one or more enzyme tryptophans.

The catalytic efficiency of 18 pig citrate synthase mutants were studied. The residues mutated were chosen according to the conservation of that residue in all known citrate synthase sequences, and the significance of that residue in substrate-amino acid interactions. Preliminary electrostatic calculations support the theory that the transition state in the rate-limiting step of the citrate synthase catalyzed reaction may be an 'enolized' version of acetyl-CoA that is neither neutral nor fully negatively charged and that a possible function for His274 is to stabilize this charge by delocalization mediated by a hydrogen bond.

The function of glutamate residue at position-217 (Glu217) in mouse adenosine deaminase (ADA) was analyzed by mutating the Glu217 active site to Asp, Gly, Gln and Ser. There were no substantial changes in secondary structure or zinc content compared to the native protein but the mutants were not able to hydrate purine riboside, a reaction caused by the wild-type enzyme to form an enzyme-inhibitor complex. Findings indicated the significance of Glu217 in catalysis.

A study of the catalytic mechanism of a single-site change in an active-site histidine residue, H320G, reveals that H320G undergo a minute change in substrate Michaelis constants (7-16)-fold and a large decrease in catalysis (600-fold). Histidine residue has been also found to interact with the oxaloacetate (OOA) carbonyl and is pivotal in its polarization. As a consequence of subunit interactions, H320G display a kinetic cooperation with CitCoA. The coordinates of this histidine residue varies by more than 5 angstrom between the closed and open form of the enzyme.

Acetyl-CoA enol acts as an intermediate in the citrate synthase 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. CS-oxaloacetate complexes with the transition-state analog inhibitor, carboxymethyl-CoA, mimic those with acetyl-CoA enol. Asp375 and His274 interact intimately with the carboxyl of the bound inhibitor. In comparison with the value of the chemical shift of the protonated carboxymethyl-CoA carboxyl in acidic aqueous solution or its value in the wild-type ternary complex, the values in the Asp375 mutants are unusually low.

Citrate synthase complexes with the transition-state analog inhibitor, carboxymethyl-CoA, are similar to those with the activated form of acetyl-CoA. The X-ray structure of the ternary complex of the enzyme, oxaloacetate and carboxymethyl-CoA has been used as the basis for a proposal that a neutral enol of acetyl-CoA is that activated form.

The rates of formation of two types of adenosine deaminase-purine riboside complexes, those which form and do not form tetrahedral intermediates, were determined. For both types, the progress curves were well-described as simple second-order approaches to equilibrium. In addition, the rate constants for adenosine association with the enzyme and tetrahedral intermediate formation are quantitatively similar. These results suggest that the rates are encounter-controlled.

A study was carried out to examine all the processes that take place in the condensation half-reaction executed by Thermoplasma acidophilum citrate synthase (TpCS). Findings revealed that the nonhydrolyzable product of the condensation reaction, dethiacitryl-CoA, forms a particularly stable complex with TpCS but not CS.

The experimental and theoretical analyses of the structural and mechanistic bases of the Thermoplasma acidophilum (TpCS) fluorescence changes that accompany the formation of the various chemical intermediates and substrate complexes with particular emphasis on the TpCS-OAA binary complex are presented. Advances in theoretical understanding tryptophan photophysics reveal that it is possible to understand changes in protein tryptophan fluorescence in some detail.

For reductions by dihydropyridines, the slopes of plots of log k vs the standard reduction potential of the dihydropyridine are not direct indicators of whether the rate-determining step is hydride-ion or hydrogen-atom transfer. When there is no substituent effect on the reverse rate, those slopes are independent of mechanism and equal to about (30 mV)-1. [ABSTRACT FROM AUTHOR]