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.
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.
Julin
D A
DA
Department of Biochemistry, University of California, Berkeley 94720.
Wiesinger
H
H
Toney
M D
MD
Kirsch
J F
JF
D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008929:Mitochondria, Heart / Q000201:enzymology*; D008962:Models, Theoretical; D009206:Myocardium / Q000201:enzymology*; D011865:Radioisotope Dilution Technique; D012997:Solvents; D013053:Spectrophotometry; D013552:Swine; D014316:Tritium
D007527:Isoenzymes; D012997:Solvents; D014316:Tritium; D001224:Aspartic Acid; D001219:Aspartate Aminotransferases