Neurospora crassa Protein Kinase: PURIFICATION, PROPERTIES, AND KINETIC MECHANISM

Abstract

Abstract A cyclic adenosine 3' : 5'-monophosphate (cyclic AMP)-independent protein kinase was purified 116-fold from cellfree extracts of Neurospora crassa mycelium. The molecular weight of the enzyme as determined by gel filtration was 60,000. The sedimentation coefficient by sucrose density gradient centrifugation was 3.8 S. The enzyme has a broad pH optimum (7.0 to 8.5). Activity was maximal at an ionic strength of 0.15. The Eact was 10.7 Cal. Kinetic studies have shown that the actual substrate for the reaction is the MgATP2- complex. Free ATP is inhibitory. The Km for MgATP2- at saturating casein is approximately 3 x 10-5 m. The Km for casein at saturating Mg-ATP2- is approximately 1.60 mg per ml. Phosvitin is the only other protein substrate tested that could replace casein. Co2+ and Mn2+ could partially substitute for Mg2+ in the reaction. Neither MgUTP2- nor MgGTP2- was a substrate. Both nucleotides were competitive inhibitors with respect to MgATP2-. Reciprocal plots of 1/v versus 1/[MgATP2-] at different fixed concentrations of casein and 1/v versus 1/[casein] at different fixed concentrations of MgATP2- were apparently parallel. However, product inhibition studies (MgADP-) and dead end inhibition studies (MgUTP2-, casein peptides) yielded results consistent with a random mechanism in which the binding of one substrate greatly decreases the affinity of the enzyme for the other substrate (Kma >> Kia, Kmb >> Kib, or in alternate terms, α >> 1).