Catalytic assessment of the glycine-rich loop of the v-Fps oncoprotein using site-directed mutagenesis.
The three glycine residues in the glycine-rich loop of the oncoprotein, v-Fps, were mutated to determine the function of these highly conserved residues in catalysis. The kinase domains of six mutants (G928A,S, G930A,S, and G933A,S) and the wild-type enzyme were expressed and purified as fusion proteins of glutathione-S-transferase in Escherichia coli, and their catalytic properties were assessed using steady-state kinetic, inhibition, viscosity and autophosphorylation studies. Although both G928A and G930A had no detectable activity toward the substrate peptide (EAEIYEAIE), the other mutants had apparent, but varying activities. G930S lowered the rate of phosphoryl transfer by 130-fold while G928S and G933S had smaller (6-9-fold) reductions in this step. These effects on catalytic function parallel the reductions in turnover and autophosphorylation but, for G933S and G933A, net product release is still rate limiting at saturating substrate and ATP concentrations. On the basis of K(I) measurements, the effects on turnover for these mutants may be due to improved ADP affinity. While ADP affinity is reduced 2- and 3-fold for G928S and G930S, the affinity of this product is increased by 22- and 7-fold for G933S and G933A. In contrast, ATP affinity is enhanced by 5-fold for G928S and G933S and is reduced by less than 2-fold for G930S. These complex, differential effects on nucleotide binding indicate that the glycines influence the relative affinities of ADP and ATP. On the basis of the results of serine replacements, Gly-928 and Gly-930 enhance ADP affinity by 9- and 2-fold compared to ATP affinity whereas Gly-933 diminishes ADP affinity by approximately 4-fold compared to ATP affinity. These findings demonstrate that the functions of the loop lie not only in modulating the rate of the phosphoryl transfer step but also in balancing the relative affinities of ATP and ADP. These effects on nucleotide specificity may be a contributing element for the stabilization of the phosphoryl transition state and may also facilitate quick release of bound products.
Hirai
T J
TJ
Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0506, USA.
Tsigelny
I
I
Adams
J A
JA
D000244:Adenosine Diphosphate / Q000494:pharmacology; D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D001667:Binding, Competitive; D002384:Catalysis; D002417:Cattle; D017124:Conserved Sequence / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism; D015681:Fusion Proteins, gag-onc / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D005998:Glycine / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D014783:Viscosity
D004791:Enzyme Inhibitors; D015681:Fusion Proteins, gag-onc; D010446:Peptide Fragments; D000244:Adenosine Diphosphate; D011505:Protein-Tyrosine Kinases; C068415:v-fps oncogene protein, Fujinami sarcoma virus; D005998:Glycine