Ribonuclease configuration

Although inversion was not observed with the E. colt alkaline phosphatase, it has been observed for ribonucleases and many other hydrolytic enzymes and for most kinases transferring phospho groups from ATP. The difference lies in the existence of a phospho-enzyme intermediate in the action of alkaline phosphatase (see Eq. 12-38). Each of the two phosphotransferase steps in the phosphatase action apparently occurs with inversion. The simplest interpretation of all the experimental results is that phosphotransferases usually act by in-line -like mechanisms which may involve metaphosphate-ion-like transition states that are constrained to react with an incoming nucleophile to give inversion. An adjacent attack with pseudorotation would probably retain the original configuration and is therefore excluded. 

Stereochemical studies support in-line mechanisms for both the transesterification and hydrolysis steps of ribonuclease catalysis. For example, chiral uridine 2, 3 -cyclic phosphorothioates are hydrolyzed with inversion of configuration, with the diastereoisomer shown yielding a 2 -monophosphothioate of the R configuration at phosphorus. 

Rennin—see chymosin Repertoire selection 415-418 Reporter group 276 Resonance Raman spectra 476 Restriction endonucleases 406-408 Restriction fragment 408 Retention of stereochemical configuration 253, 254 Reverse genetic s 438 -442 Reverse transcriptase 3 8 Rhizopus-pepsin 486, 490 Ribonuclease A 9... 

The first chiral phosphates to be used for stereochemical analyses were chiral phosphorothioates, which were used to determine the stereochemical courses of ribonuclease, UDP-glucose pyrophosphorylase, adenylate kinase and several other kinases and synthetases. The chiral phosphorothioates either had sulfur in place of an oxygen at an otherwise prochiral center of a phosphodiester or phosphoanhydride or stereospecifically placed sulfur and 180 (or nO) in a terminal phosphoryl group. The syntheses and configurational analyses of the most important of these compounds are outlined in the following. 

Among all possible configurations, the active enzyme conformation is assumed to be thermodynamically the most stable in situ. Experimental evidence to validate this assumption came from reversible denaturation studies on enzymes. For example. ribonuclease may be completely unfolded in urea solution after breakage of four native intramolecular disulphide bridges. Afterwards, when ribonuclease is brought to renature in the proper conditions, the native disulphide bonds are restored, and the protein recovers its catalytic activity. This case must be regarded, however, as a very favourable one because in most instances renaturation is... 

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