IDENTIFICATION OF ACTIVE-SITE HISTIDINE-RESIDUES OF A SELF-INCOMPATIBILITY RIBONUCLEASE FROM A WILD TOMATO

The style component of the self-incompatibility (S) locus of the wild tomato Lycopersicon peruvianum (L.) Mill. is an allelic series of glyc oproteins with ribonuclease activity (S-RNases). Treatment of the S-3- RNase from L. peruvianum with iodoacetate at pH 6.1 led to a loss of R Nase activity. In the presence of a competitive inhibitor, guanosine 3 '-monophosphate (3'-GMP), the rate of RNase inactivation by iodoacetat e was reduced significantly. Analysis of the tryptic digestion product s of the iodoacetate-modified S-RNase by reversed-phase high-performan ce liquid chromatography and electrospray-ionization mass spectrometry showed that histidine-32 was preferentially modified in the absence o f 3'-GMP. Histidine-88 was also modified, but this occurred both in th e presence and absence of 3'-GMP, suggesting that this residue is acce ssible when 3'-GMP is in the active site. Cysteine-150 was modified by iodoacetate in the absence of 3'-GMP and, to a lesser extent, in its presence. The results are discussed with respect to the related fungal RNase T-2 family and the mechanism of S-RNase action.