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 glycoproteins with ribonuclease activity (S-RNases). Treatment of the S3-RNase from L. peruvianum with iodoacetate at pH 6.1 led to a loss of RNase activity. In the presence of a competitive inhibitor, guanosine 3'-monophosphate (3'-GMP), the rate of RNase inactivation by iodoacetate was reduced significantly. Analysis of the tryptic digestion products of the iodoacetate-modified S-RNase by reversed-phase high-performance liquid chromatography and electrospray-ionization mass spectrometry showed that histidine-32 was preferentially modified in the absence of 3'-GMP. Histidine-88 was also modified, but this occurred both in the presence and absence of 3'-GMP, suggesting that this residue is accessible 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 T2 family and the mechanism of S-RNase action.
Parry
S
S
Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, Victoria, Australia.
Newbigin
E
E
Currie
G
G
Bacic
A
A
Oxley
D
D
D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000494:pharmacology; D006639:Histidine; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D018551:Solanum lycopersicum / Q000201:enzymology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D012260:Ribonucleases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D006023:Glycoproteins; D007461:Iodoacetates; D010446:Peptide Fragments; D006639:Histidine; D006157:Guanosine Monophosphate; D012260:Ribonucleases; C022542:ribonuclease S; D019807:Iodoacetic Acid