Kinetic isotope effect studies on aspartate aminotransferase: evidence for a concerted 1,3 prototropic shift mechanism for the cytoplasmic isozyme and L-aspartate and dichotomy in mechanism

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Abstract
The C.alpha. primary hydrogen kinetic isotope effects (C.alpha.-KIEs) for the reactions of the cytoplasmic isozyme of aspartate aminotransferase (cAATase) with [.alpha.-2H]-L-aspartate are small and only slightly affected by deuterium oxide solvent (DV = 1.43 .+-. 0.03 and DV/KAsp = 1.36 .+-. 0.04 in H2O; DV = 1.44 .+-. 0.01 and DV/KAsp = 1.61 .+-. 0.06 in D2O). The solvent KIEs (SKIEs) are somewhat larger and are essentially independent of deuterium at C.alpha. (D2OV = 2.21 .+-. 0.07 and D2OV/KAsp = 1.70 .+-. 0.03 with [.alpha.-1H-aspartate; D2OV = 2,34 .+-. 0.12 and D2OV/KAsp = 1.82 .+-. 0.06 with [.alpha.-2H]-L-aspartate). The C.alpha.-KIEs on V and on V/KAsp are independent of pH from 5.0 to pH 10.0. These results support a rate-determining concerted 1.3 prototropic shift mechanism by the multiple KIE criteria [Hermes, J. D., Roeske, C. A., O''Leary, M. H., and Cleland, W. W. (1982) Biochemistry 21, 5106] The large C.alpha.-KIEs for the reaction of mitocyhondrial AATase (mAATase) with L-glutamate (DV = 1.88 .+-. 0.13 and DV/KGlu = 3.80 .+-. 0.43 in H2O; DV = 1.57 .+-. 0.05 and DV/KGLu = 4.21 .+-. 0.19 in D2O) coupled with the relatively small SKIEs (D2OV = 1.58 .+-. 0.04 and D2OV/KGlu = 1.25 .+-. 0.05 with [.alpha.-1H]-L-glutamate; D2OV = 1.46 .+-. 0.06 and D2OV/KGlu = 1.16 .+-. 0.05 with [.alpha.-2H]-L-glutamate) are most consistent with a two-step mechanism for the 1,3 prototropic shift for this isozyme-substrate pair. Primary C.alpha.-hydrogen and SKIEs on the mAATase plus L-aspartate, cAATase plus L-glutamate, and cAATase plus L-alanine reactions are consistent with either a one- or two-step mechanism. Solvent isotope effects on the competitive inhibition constants for maleate and .alpha.-methyl-D,L-aspartate are "D2OKi = 1.35 .+-. 0.06 and 1.11 .+-. 0.03, respectively. The KIEs together with the previous results [Julin, D. A., Wiesinger, H., Toney, M. D., and Kirsch, J. F. (1989) Biochemistry (first of three papers in this issue); McLeish, M. J., Julin, D. A. and Kirsch, J. f. (1989) Biochemistry (second of three papers in this issue)] provide the basis for the partial construction of free energy profiles for these reactions.