SOURCE OF RESPONSE REGULATOR AUTOPHOSPHATASE ACTIVITY - THE CRITICAL ROLE OF A RESIDUE ADJACENT TO THE SPOOF AUTOPHOSPHORYLATION ACTIVE-SITE

Two-component signaling systems are used by bacteria, plants, and lowe r eukaryotes to adapt to environmental changes. The first component, a protein kinase, responds to a signal by phosphorylating the second co mponent; a response regulator protein that often acts by inducing the expression of specific genes. Response regulators also have an autopho sphatase activity that ensures that the proteins are not permanently a ctivated by phosphorylation. The magnitude of this activity varies by at least 1000-fold between various response regulators, and the molecu lar features responsible for this varied autophosphatase activity have not been clearly defined. Using wild-type and mutant derivatives of t he sporulation response regulator SpoOF, it has been demonstrated that a key residue in determining the magnitude of this activity is that a t position 56 of SpoOF similar to P; this residue is adjacent to the s ite of phosphorylation, Asp 54. For example, SpoOF similar to P K56N h as a 23-fold greater autophosphatase activity (t(1/2) = 8 min) than wi ld-type SpoOF similar to P (t(1/2) = 180 min). It is suggested that, b y analogy to the GTPase activity of p21(ras) and by examining the crys tallographic structure of SpoOF, that the carboxyamide of the mutant A sn 56 may favorably position a catalytic water near the protein acyl p hosphate to promote SpoOF similar to P K56N hydrolysis. It is also ded uced that Lys 56 in the wild-type protein is critical for the efficien t interaction and phosphoryl transfer between SpoOF and it's cognate p rotein kinase, KinA. Comparison of the known response regulators shows that inefficient autophosphatases (t(1/2) on the order of hours) typi cally contain an amino acid residue with a long side chain at the posi tion equivalent to 56 in SpoOF, whereas efficient autophosphatases (t( 1/2) on the order of minutes) frequently contain a residue with a carb oxyamide or carboxylate side chain at this position. It appears that, by altering residues adjacent to the active site, the autophosphatase activity of response regulator proteins has been attenuated to match t he diverse biological roles played by these proteins.