PLANT PHYSIOLOGY , Vol 115, Issue 4 1421-1429, Copyright © 1997 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Identification of Active-Site Histidine Residues of a Self-Incompatibility Ribonuclease from a Wild Tomato
S. Parry, E. Newbigin, G. Currie, A. Bacic and D. Oxley
Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, Victoria 3052, Australia
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[prime]-monophosphate (3[prime]-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[prime]-GMP. Histidine-88 was also modified, but this occurred
both in the presence and absence of 3[prime]-GMP, suggesting that this
residue is accessible when 3[prime]-GMP is in the active site. Cysteine-150
was modified by iodoacetate in the absence of 3[prime]-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.
