Specificity of natural and artificial substrates for human Cdc25A

Cdc25A is a dual-specific protein phosphatase involved in the regulation of the kinase activity of Cdk-cyclin complexes in the eukaryotic cell cycle. To understand the mechanism of this important regulator, we have generated highly purified biochemical reagents to determine the kinetic constants for human Cdc25A with respect to a set of peptidic, artificial, and natural su bstrates, Cdc25A and its catalytic domain (dN25A) demonstrate very similar kinetics toward the artificial substrates p-nitrophenyl phosphate (k(cat)/K -m 15-25 M-1 s(-1)) and 3-O-methylfluorescein phosphate (k(cat)/K-m = 1.1-1 .3 x 10(4) M-1 s(-1)). Phospho-peptide substrates exhibit extremely low sec ond-order rate constants and a flat specificity profile toward Cdc25A and d N25A (k(cat)/K-m = 1 to 10 M-1 s(-1)). In contrast to peptidic substrates, Cdc25A and dN25A are highly active phosphatases toward the natural substrat e, T14- and Y15-bis-phosphorylated Cdk2/CycA complex (Cdk2-pTpY/ CycA) with k(cat)/K-m values of 1.0-1.1 x 10(6) M-1 s(-1). In the context of the Cdk2 -pTpY/CycA complex, phosphothreonine is preferred over phospho-tyrosine by more than 10-fold. The highly homologous catalytic domain of Cdc25c is esse ntially inactive toward Cdk2-pTpY/CycA. Taken together these data indicate that a significant degree of the specificity of Cdc25 toward its Cdk substr ate resides within the catalytic domain itself and yet is in a region(s) th at is outside the phosphate binding site of the enzyme. (C) 2001 Academic P ress.