Evidence for kinetically distinct forms of pp60c-src with different Km values for their protein substrate.

The biphasic kinetics for phosphorylation of poly(E4Y) by the protein tyrosine kinase pp60c-src were examined. At pH 6.5 substrate inhibition was observed, whereas at pH 8.0 the kinetics were still biphasic, but the enzyme was no longer inhibited. The reaction rate increased in a nonlinear fashion with increasing concentration of substrate. The kinetics were examined from the view that the biphasic kinetics at pH 8.0 were due to two enzymes acting simultaneously on the same substrate. A 55-fold difference in Km values (0.029 versus 1.6 mg/ml) was calculated. The low Km form of the enzyme (0.043 mg/ml) was physically separated from the mixture of kinetic variants by immunoaffinity chromatography, and phosphorylation by protein kinase A resulted in the formation of an enzyme with an intermediate Km (0.3-0.4 mg/ml). The presence of multiple kinetic forms of this tyrosine kinase has important implications in our efforts to understand the role of pp60c-src in human oncology.