Structural features facilitating the glutamate dehydrogenase catalyzed α-imino acid-α-amino acid interconversion

Abstract

Glutamate dehydrogenase catalyzes the reversible oxidation of l-proline and l-pi-pecolic acid to the corresponding cyclic α-imino acids. The active substrates are the amino acid anion in one direction and the iminium ion in the other. The oxidation of the ester, amide, and N-methyl derivatives of l-proline by enzyme-NADP + and the reduction of N-methyl-Δ 1-tetrahydropyridinium ion by enzyme-NADPH do not proceed to a detectable extent under the experimental conditions. The methyliminium ion, however, undergoes facile nonenzymatic reduction by NADPH. If it is assumed that the nonenzymatic reaction reflects the structural requirements of the redox step of the enzymatic reaction, then the lack of reactivity of the tetrahydropyridinium ion toward enzyme-NADPH must be due to the instability of the central complex. It appears that the α-carboxylate and NH groups in the amino acid anion and in the α-imino acid are involved in binding the substrates to the enzyme-coenzyme complexes. We conclude that each of these active substrates binds to its appropriate enzyme-coenzyme complex through a hydrogen bond between its NH group and a basic enzyme group; there is also an ionic bond in the central complex between the α-carboxylate group of the active substrate and a positively charged enzyme group. The five-membered amino acid anions are more reactive toward enzyme-NADP + than the six-membered ones. The same reactivity order is seen for the reduction of imino acids by enzyme-NADPH. Since these effects are also present in the nonenzymatic reduction by NADPH we ascribe the ring size effects on V K substrate primarily to those on the hydride-transfer step.