Hybridization of glutamate aspartate transaminase. Subunit interaction

Abstract

Glutamate aspartate transaminase (EC 2.6.1.1) is a dimeric enzyme with identical subunits with each active site containing pyridoxal 5'-phosphate linked via an internal Shiff's base to a lysine residue. It is not known if these sites interact during catalysis but negative cooperativity has been reported for binding of the coenzyme (Arrio-Dupont, M. (1972), Eur. J. Biochem. 30, 307). Also nonequivalence of its subunits in binding 8-anilinonaphthalene-1-sulfonate (Harris, H.E., and Bayley, P. M. (1975), Biochem. J. 145, 125), in modification of only a single tyrosine with full loss of activity (Christen, P., and Riordan, J.F. (1970), Biochemistry 9, 3025), and following modification with 5,5'-dithiobis(2-nitrobenzoic acid) (Cournil, I., and Arrio-Dupont, M. (1973), Biochemie 55, 103) has been reported. However, steady-state and transient kinetic methods as well as direct titration of the active site chromophore with substrates and substrate analogs have not revealed any cooperative phenomena (Braunstein, A. E. (1973), Enzymes, 3rd Ed. 9, 379). It was therefore decided that a more direct approach should be used to clarify the quistion of subunit interaction during the covalent phase of catalysis. To this end a hybrid method was devised in which a hybrid transaminase was prepared which contained one subunit with a functional active site while the other subunit has the internal Shiff's base reduced with NaBH4. The specific activities and amount of "actively bound" pyridoxal 5'-phosphate are both in a 2:1 ratio for the native and hybrid forms. Comparison of the steady-state kinetic properties of the hybrid and native enzyme forms shows that both forms gave parallel double reciprocal plots which is characteristic of the Ping-Pong Bi-Bi mechanism of transamination. The Km values for the substrates L-aspartic acid and alpha-ketoglutaric acid are nearly identical while the Vmax value for the hybrid is one-half the value of the native transaminase. It therefore appears that the active sites of glutamate aspartate transaminase function independently and a compulsory flip-flop mechanism is not involved.

Refolding Intermediates of Acid-unfolded Mitochondrial Aspartate Aminotransferase Bind to hsp70

“…1A). We are assuming that activity is recovered only in the final phase of folding which agrees with the fact that only the fully folded native dimer of pmAAT is active (25,40). In the mathematical treatment used to analyze Reaction 1 (41), irreversibility only implies that the reverse reactions have rate constants much smaller than those for the forward reactions.…”

Engineering homooligomeric proteins to detect weak intersite allosteric communication: Aminotransferases, a case study

“…The last 40 years has witnessed several conflicting reports on whether other AATases are cooperative enzymes [19][20][21][22][23][24][25][26] ; however the presence of allosteric communication between the active sites of eAATase has not been reported. This controversy has also arisen with other aminotransferases such as glutamate-1-semialdehyde aminotransferase for which crystal structures from Synechococcus 27 and Bacillus subtilis 28 show asymmetric and symmetric active sites, respectively.…”

Biological Magnetic Resonance

“…The system consists of two groups of chemically and immunologically distinct proteins, each possessing multiple forms with similar properties (Martinez-Carrion and Tiemeier, 1967;Martinez-Carrion et al, 1970). Two monomers of 47,000 molecular weight are associated to form the active enzyme and it appears from hybridization experiments that the active sites function independently (Boettcher and Martinez-Carrion, 1975). The enzyme requires pyridoxal-5f-phosphate as a cofactor.…”