Estimation of free energy barriers in the cytoplasmic and mitochondrial aspartate aminotransferase reactions probed by hydrogen-exchange kinetics of C.alpha.-labeled amino acids with solvent

Abstract

The existence of the postulated quinonoid intermediate in the cytoplasmic aspartate amino-transferase catalyzed transamination of aspartate to oxaloacetate was probed by determining the extent of transfer of tritium from the C alpha position of tritiated L-aspartate to pyridoxamine 5'-phosphate in single turnover experiments in which washout from the back-reaction was obviated by product trapping. The maximum amount of transferred tritium observed was 0.7%, consistent either with a mechanism in which a fraction of the net transamination reaction proceeds through a quinonoid intermediate or with a mechanism in which this intermediate is formed off the main reaction pathway. It is shown that transfer of labeled hydrogen from the amino acid to cofactor cannot be used to differentiate a stepwise from a concerted transamination mechanism. The amount of tritium transferred is a function of the rate constant for torsional equilibration about the epsilon-amino group of Lys-258, the presumptive abstractor of the C alpha proton; the relative rate constants for hydrogen exchange with solvent versus cofactor protonation; and the tritium isotope effect on this ratio. The free energy barriers facing the covalent intermediate between aldimine and keto acid product (i.e., ketimine and possibly quinonoid) were evaluated relatively by comparing the rates of C alpha-hydrogen exchange in starting amino acid with the rates of keto acid formation. The value of theta (= kexge/kprod) was found to be 2.6 for the reaction of cytoplasmic isozyme with aspartate and ca. 0.5 for that of the mitochondrial form with glutamate.

Structural Basis for the Inhibition of the Biosynthesis of Biotin by the Antibiotic Amiclenomycin

“…The same residue would then be able to transfer a proton to the C4Ј atom of PLP. In these proton transfer steps Lys-274 acts in the same manner as in the classical mechanism for aminotransferases (37,38). To complete the aromatization of the cyclohexadiene ring, the proton at the C1 carbon of the ring has to be removed.…”

Reengineering the catalytic lysine of aspartate aminotransferase by chemical elaboration of a genetically introduced cysteine

“…The properties of the inactive K258C mutant and of the chemically elaborated enzyme are reported. Julin and were prepared as described in Julin et al (1989). All buffer and reagent solutions used for chemical modifications and enzyme resolutions were degassed prior to use.…”

Aspartate aminotransferase catalyzed oxygen exchange with solvent from oxygen-18-enriched .alpha.-ketoglutarate: evidence for slow exchange of enzyme-bound water

“…The kinetic constants for the m A AT ase-catalyzed reaction of L-aspartate and aKG in 25 mM, pH 8.3 TAPS buffer at 10 °C were determined to be 51 ± 3 s-1,0.35 ± 0.05 mM, and 0.32 ± 0.05 mM for &£at, KAsp, and KoKG, respectively. The corresponding constants at 25 °C are 255 s-1, 0.52 mM, and 0.57 mM (Julin & Kirsch, 1989). The results at 10 °C were obtained at a ratio of 10 units of MDH per unit of mAATase.…”

“…The relative energy barriers facing the first intermediate formed upon labilization of the Ca proton from the a-amino acid in the aspartate aminotransferase (AATase)1 reaction were measured by monitoring the relative rates of Ca-deu-terium exchange with solvent and of -keto acid formation (Julin et al, 1989). This paper describes a probe of the barriers for the reverse AATase reaction (aKG plus the pyridoxamine S'-phosphate form of the enzyme) by comparing the rate of exchange of oxygen-18 from carbonyl-lsO-enriched 1 Abbreviations: TAPS, 3- [[tris(hydroxymethyl)methyl]amino]propanesulfonic acid; aKG, a-ketoglutarate; 2,4-DNPH, 2,4-dinitrophenylhydrazine; MDH, malic dehydrogenase; mAATase, mitochondrial aspartate aminotransferase.…”

“…The specific activity of the mAATase was determined to be approximately 230 units/mg of protein (Julin & Kirsch, 1989). The reaction was assayed in the direction of Lglutamate and oxaloacetate production by coupling to the MDH reaction (Velick & Vavra, 1962) and monitoring the decrease in the absorbance of NADH at 340 or 389.7 nm («389.7 = 454 M"1 cm"1)• The values of Km and Kmax at 10 °C were determined in reaction mixtures containing 25 mM TAPS buffer, 0.2 mM NADH, and 2.86 units/mL MDH (186 nM).…”