| Abstract
| - Based on the X-ray structure of the insulin receptor kinase [Hubbard, S. R. (1997) EMBO J.16, 5572−5581], Arg-1130 in the oncoprotein v-Fps, a nonreceptor tyrosine protein kinase, is predictedto interact with the P+1 glutamate in substrate peptides. To determine whether this residue is an importantrecognition element in v-Fps, Arg-1130 was substituted with leucine (R1130L) and glutamic acid (R1130E).The ability of these mutants to phosphorylate the peptide EAEIYXAIE, where X is glutamic acid, alanine,or lysine, was assessed. A comparison of the rates of peptide phosphorylation under limiting substrateconcentrations (i.e., kcat/Km conditions) indicates that substrate specificity is altered by the electrostaticenvironment of the P+1 pocket. When the pocket displays a positive charge (Arg-1130; wild type), nocharge (R1130L), or a negative charge (R1130E), v-Fps prefers to phosphorylate the glutamate peptideover the lysine peptide by a 200:1, 9:1, or 1:1 margin. While kcat/Km for the glutamate peptide is 50-foldhigher for wild type compared to R1130E, kcat/Km for the lysine peptide is 3-fold higher for R1130Ecompared to wild type, a 150-fold change in relative substrate specificity. Analysis of the individual stepsin the kinetic mechanism using viscosometric techniques indicates that the wild-type enzyme binds theglutamate peptide 3-fold better than the alanine peptide and, at least, 10-fold better than the lysine peptide.For R1130L, this margin range is reduced substantially, and for R1130E, no binding preference is observed.Nonetheless, the lysine peptide binds, at least, 4-fold better to R1130E than to wild type, and the glutamatepeptide binds 3-fold poorer to R1130E than to wild type. The mutants lower the phosphoryl transfer rateby 4−30-fold for the three peptides, suggesting that Arg-1130 helps to position the tyrosine for optimumcatalysis. The data indicate that a single mutation in v-Fps can alter significantly the relative substratespecificity by about 2 orders of magnitude with, at least, 50% of this effect occurring through relativechanges in peptide binding affinity.
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