Kinetic mechanism and ATP-binding site reactivity of p38 gamma MAP kinase

Activated p38 gamma MAP kinase exhibited significant basal ATPase activity in the absence of a kinase substrate, and addition of a phosphoacceptor sub strate increased k(cat)/K-m > 20-fold. AMP-PCP was competitive with ATP bin ding and noncompetitive with phosphoacceptor substrate binding. The nucleot ide binding site affinity label 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA ) bound stoichiometrically at Lys-56 in the ATP site of both unphosphorylat ed and activated p38 gamma, AMP-PCP only protected the activated enzyme fro m FSBA inactivation, implying that AMP-PCP does not bind unphosphorylated p 38 gamma. Basal ATPase activities were also observed for activated p38 alph a, ERK2 and JNK3 suggesting that the enzymatic mechanism may be similar for all classes of MAP kinases, (C) 1999 Federation of European Biochemical So cieties.