ON THE MECHANISM OF ACTION OF RIBONUCLEASE-A - RELEVANCE OF ENZYMATICSTUDIES WITH A P-NITROPHENYLPHOSPHATE ESTER AND A THIOPHOSPHATE ESTER

It has been reported that His-119 of ribonuclease A plays a major role as an imidazolium ion acid catalyst in the cyclization/cleavage of no rmal dinucleotides but that it is not needed for the cyclization/cleav age of 3'-uridyl p-nitrophenyl phosphate. We see that this is also tru e for simple buffer catalysis, where imidazole (as in His-12 of the en zyme), but not imidazolium ion, plays a significant catalytic role wit h the nitrophenyl substrate, but both are catalytic for normal dinucle otides such as uridyluridine. Rate studies show that the enzyme cataly zes the cyclization of the nitrophenylphosphate derivative 47,000,000 times less effectively (k(cat)/k(uncat)) than it does uridyladenosine, indicating that approximate to 50% of the catalytic free energy chang e is lost with this substrate. This suggests that the nitrophenyl subs trate is not correctly bound to take full advantage of the catalytic g roups of the enzyme and is thus not a good guide to the mechanism used by normal nucleotides. The published data on kinetic effects with rib onuclease A of substituting thiophosphate groups for the phosphate gro ups of normal substrates has been discussed elsewhere, and it was argu ed that these effects are suggestive of the classical mechanism for ri bonuclease action, not the novel mechanism we have recently proposed. The details of these rate effects, including stereochemical preference s in the thiophosphate series, can be invoked as support for our newer mechanism.