Isolation and characterization of mutated mitochondrial GTP:AMP phosphotransferase.
GTP:AMP phosphotransferase (adenylate kinase isozyme 3, AK3) mutants were obtained by using the ability of AK3 to complement a temperature-sensitive mutation of Escherichia coli adenylate kinase (AKe). Five mutants, P16L, G19S, G91D, G91S, and P93L, had mutation sites located at two loops that are involved in substrate binding of the enzyme. P16L and G19S bearing changes at the first loop showed reduced affinity for both GTP and AMP, the extent of reduction being slightly higher for GTP than AMP. In 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 Vmax value of the enzyme. Another mutant, D163N, had a substitution at the site forming a salt bridge in adenylate kinase isozyme 1 (AK1), which influenced the Vmax as well as the Km values for both substrates. The kinetic characteristics of these mutants were comparable to those of the corresponding AK1 or AKe mutants. Furthermore, from the results of mutations T201P and T201A that had alterations in all the kinetic parameters of AK3 and from a comparison with the structure and the kinetic parameters of AKe, we expect that a residue(s) around Thr201 is involved in recognition of the base of nucleoside triphosphate.
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, 753-8515, Japan.
M
M
Yamada
M
M
Sugahara
Y
Y
Hishitani
M
M
Nobumoto
A
A
Nakazawa
D000263:Adenylate Kinase / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D009154:Mutation / Q000235:genetics; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D007527:Isoenzymes; D006160:Guanosine Triphosphate; C021894:nucleoside triphosphate-adenylate kinase; D000263:Adenylate Kinase; C110939:adenylate kinase 1; D009703:Nucleoside-Phosphate Kinase