Glutamic-Aspartic Transaminase: X. MECHANISM AND ORDER OF FORMATION OF THE ENZYME-SUBSTRATE CARBOXYLATE BONDS

Abstract

Abstract Glutarate ion is bound to the pig heart extramitochondrial glutamic-aspartic transaminase (EC 2.6.1.1) by both carboxylate groups at low pH values but by only one carboxylate group at high pH values. The positively charged protein site which binds a carboxylate group at both high and low pH values is normally masked by the buffer anion. A spectroscopic method is presented for studying the affinity of this one site on the protein for any anion. Although there is little free energy and no enthalpy change in the equilibrium, glutarate cannot displace this buffer anion directly at high pH values but does so by a series of reactions involving the acidic glutarate complex which has both carboxylate groups bound. The site at which the second carboxylate group is bound at low pH values is thought to be the quaternary nitrogen atom corresponding to the e-amino group of a lysine residue involved in binding the aldehyde group of the chromophoric prosthetic group, pyridoxal phosphate. It is suggested that the binding of substrate anions to proteins in other enzymatic reactions does not necessarily involve the unmasking of a positive site by a conformational change but commonly involves displacement either of the buffer ion or the reaction product. Experimental methods are presented to test this hypothesis.