A CRYSTALLOGRAPHIC REINVESTIGATION INTO THE STRUCTURE OF STREPTOMYCES-GRISEUS PROTEINASE-A REVEALS AN ACYL-ENZYME INTERMEDIATE

A molecular dynamics (MD) simulation on the contents of two asymmetric units of the crystal structure of the bacterial serine proteinase, St reptomyces griseus proteinase A (SGPA), resulted in four dihydrogen ph osphate anions migrating to form a cluster in a solvent region far rem oved from the protein surface. In an effort to provide experimental ve rification for this unexpected phenomenon, native SGPA crystals were s oaked in 2.0 M KH2AsO4; intensity data were collected and difference e lectron density maps examined for evidence of H2AsO4- ion clusters. Th ese maps did not corroborate the cluster observed in the MD simulation . They did, however, show positive electron density features located i n the active site cavity that could be interpreted as a tetrapeptide, Gly-Ala-Ser-(beta-OH)Asp, covalently bonded to O-gamma of Ser195 as an acyl-enzyme intermediate. There was also a peak of electron density c orresponding to an ideal position for the hydrolytic water in the deac ylation reaction. This density is centred 3.1 Angstrom from His57 N-ep silon 2 and 2.7 Angstrom from the carbonyl-carbon atom of the planar a cyl-ester bond of the complex. The carbonyl-oxygen atom of the ester i s located in the oxyanion pocket and participates in hydrogen bends wi th Gly193 NH (2.6 Angstrom) and Ser195 NH (3.0 Angstrom).