IDENTIFICATION OF HISTIDINE-31 AND CYSTEINE-95 IN THE ACTIVE-SITE OF SELF-INCOMPATIBILITY ASSOCIATED S-6-RNASE IN NICOTIANA-ALATA

S-RNase is associated with the gametophytic self-incompatibility of fl owering plants in Solanaceae and, on the basis of sequence homology, b elongs to the RNase T-2 family. To identify the active site residues i n S-RNase, Nicotiana alata S-6-RNase was studied by chemical modificat ion. S-6-RNase was inactivated with iodoacetic acid under conditions s imilar to those used for the inactivation of RNase T-2. No inactivatio n took place in the presence of 2' GMP. Analysis of carboxymethylated S-6-RNase revealed that the S-carboxymethylation of Cys95 caused inact ivation of the enzyme and that the two histidine residues correspondin g to two essential histidine residues of RNase T-2 remained intact. Tr eatment of S-6-RNase with diethyl pyrocarbonate (DEPC) resulted in los s of enzyme activity, and the enzyme was protected from inactivation i n the presence of 2' GMP. The ethoxycarbonylated residues in DEPC-inac tivated S-6-RNase were analyzed by mass spectrometry, which also provi ded structural information on sugar moieties attached to Asn27 and Asn 37, His31 was modified with DEPC in the absence of 2' GMP and was not modified in its presence, His31 and His91 are conserved in all members of the RNase T-2 family sequenced so far, but Cys95 is not conserved in all known Solanaceae S-RNases, These results suggest that His31, po ssibly together with His98, corresponding to His115 at the active site of RNase T-2, is essential to the function of S-6-RNase, but Cys95 is not essential though its S-carboxymethylation causes inactivation.