ISOLATION AND CHARACTERIZATION OF MUTATED MITOCHONDRIAL GTP-AMP PHOSPHOTRANSFERASE

GTP:AMP phosphotransferase (adenylate kinase isozyme 3, AK3) mutants w ere obtained by using the ability of AK3 to complement a temperature-s ensitive mutation of Escherichia coli adenylate kinase (AKe). Five mut ants, P16L, G19S, G91D, G91S, and P93L, had mutation sites located at two loops that are involved in substrate binding of the enzyme. P16L a nd G19S bearing changes at the first loop showed reduced affinity for both GTP and AMP, the extent of reduction being slightly higher for Gm than AMP. Ln contrast, G91S and P93L having alterations at the second loop had lower affinities for AMP. Only the alterations at the second loop strongly influenced the V-max value of the enzyme. Another mutan t, D163N, had a substitution at the site forming a salt bridge in aden ylate kinase isozyme 1 (AK1), which influenced the V-max as well as th e K-m values for both substrates. The kinetic characteristics of these mutants were comparable to those of the corresponding AK1 or AKe muta nts. Furthermore, from the results of mutations T201P and T201A that h ad alterations in all the kinetic parameters of AK3 and from a compari son with the structure and the kinetic parameters of AKe, we expect th at a residue(s) around Thr201 is involved in recognition of the base o f nucleoside triphosphate. (C) 1998 Academic Press.