Ribonuclease transition state complex

The association of bamase, an extracellular ribonuclease, with its intracellular inhibitor, barstar, provides a particularly well-characterized example of electrostatically steered protein-protein encounter. The association rate is very fast (about 10 -10 at 50 mM ionic strength), and mutation and ionic-strength-dependence studies clearly show the influence of electrostatic interactions. Brownian dynamics simulations are able to reproduce the ionic-strength dependence of the rate for the wild-type proteins and the rates for wild-type and 11 mutants at 50 mM ionic strength to within a factor of 2. These simulations provide insight into the structure of the encounter (transition state) complex in which barstar tends to be shifted from its position in the bound complex towards the guanine binding loop on bamase. 

In recent years attention has focused on the role of intrinsic binding energy and entropic factors as major contributors to enzyme catalytic efficiency (Page and Jencks, 197l Jencks, 1975,1981). The ribonuclease mechanism conforms to expectations based on these ideas. In particular, distortion occurs to raise the ground state of the substrate in the S complex, and the bound substrate interacts with the enzyme in a manner such that the enzyme becomes complementary to the transition state of the reaction during the catalytic cycle. 

The preceding summary and Fig. 20 present a frame-by-frame account of the pathway for ribonuclease catalysis, based predominandy on knowledge of the structures of the various intermediates and transition states involved. The ability to carry out such a study is dependent on three critical features (1) crystals of the enzyme which diffract sufficiently well to permit structural resolution to at least 2 A (2) compatibility of the enzyme, its crystals, and its catalytic kinetic parameters with cryoenzymology so as to permit the accumulation and stabilization of enzyme-substrate complexes and intermediates at subzero temperatures in fluid cryosolvents with crystalline enzyme and (3) the availability of suitable transition state analogs to mimic the actual transition states which are, of course, inaccessible due to their very short lifetimes. The results from this investigation demonstrate that this approach is feasible and can provide unparalleled information about an enzyme at work. 

Vanadate itself is a much poorer inhibitor of ribonuclease A than is the VUr complex. This makes sense because vanadate alone cannot mimic the transition state. Covalently bound components, the parts of the nucleoside covalently bound to the phosphate moiety, are needed to complete the transition-state-like structure. They... 

Leon-Lai, C.H., M.J. Gresser, and A.S. Tracey. 1996. Influence of vanadium(V) complexes on the catalytic activity of ribonuclease A. The role of vanadate complexes as transition state analogues to reactions at phosphate. Can. J. Chem. 74 38 -8. 

Ray, W.J., Jr. and C.B. Post. 1990. The oxyvanadium constellation in transition-state-analogue complexes of phosphoglucomutase and ribonuclease. Structural deductions from electron-transfer spectra. J. Biochem. 29 2779-2789. 

Borah, B., C.W. Chen, W. Egan, M. Miller, A. Wlodawer, and J.S. Cohen. 1985. Nuclear magnetic resonance and neutron diffraction studies of the complex of ribonuclease A with uridine vanadate, a transition-state analogue. J. Biochem. 24 2058-2067. 

Deng, H., J.W. Burgner, II, and R.H. Callender. 1998. Structure of the ribonuclease A-uridine-vanadate transition state analogue complex by Raman difference spectroscopy Mechanistic implications. J. Am. Chem. Soc. 120 4717-4722. 

Lindquist, R.N. Lyon, J.L. Lienhard, G.E. Possible transition-state analogs for ribonuclease, the complexes of uridine with oxyvanadium (IV) ion and vanadium (V) ion. J, Amer. Chem. Soc., 1973, 95, 8762-8768. 

Diamino-2 ,3 -dideoxyadenosine has been converted into the water-stable technetium complex 64, as a model for the transition state in ribonuclease- catalysed hydrolysis of inter-nucleotidic links. 

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