Structural aspects of protein catalysis

Abstract

The results presented in this thesis deal with the studies on the structure and activity changes of enzyme Ribonuclease-A during the initial stages of denaturation in highly acidic aqueous solutions, with special reference to the isolation and structural characterisation of an enzymatically active intermediate. When RNase A was maintained in dilute solutions of HCl or H?SO? for different intervals of time at 30°C and the products analysed at neutral pH, it was observed that the enzyme was relatively stable towards changes in activity in 0.1N acid. However, in 0.5M HCl (or 0.5M H?SO?) the enzyme was found to retain its original activity only for a period of 10 hours; subsequently there was gradual loss in activity. At still higher concentrations of the acid, the initial lag period was reduced and the rate of loss of activity was considerably increased. It was also found that RNase A in 0.5M HCl does not exhibit any changes in its ultraviolet absorption spectrum for a period of 22 hours. The activity changes of RNase A under acidic conditions have been found to be dependent on the enzyme concentration. Activity determinations of the reaction products in presence of S-protein and S-peptide indicated structural modifications in the S-protein region, during the initial stages of acid denaturation. For studies on the structural changes associated with acid denaturation, a protein concentration of 10??M and an acid concentration of 0.5M at 30°C was generally employed. N-terminal end group analysis of the HCl-reaction products revealed that neither peptide bond cleavage nor N-acyl shift occurs at least up to 60 hours of reaction. Cleavage at peptide linkages were apparent only at later stages of the reaction. A 60?hour reaction product was found to have the same amino acid composition as that of RNase A. By electrophoretic studies, however, this product was found to have a uniform distribution of charge and a cathodic mobility less than that of RNase A. It was observed that ammonia is released from the beginning of the reaction, indicating that deamidination of RNase A is a primary reaction in the initial stages of acid denaturation. The release of ammonia was found to have an initial fast rate for a period of 5 hours (during which time nearly 0.5 mole of ammonia per mole of protein was released), a markedly slow rate for the next 10 hours and a uniform fast rate during the subsequent period. The lag period after the initial release of 0.5 mole of ammonia could most reasonably be attributed to the resistance of a monodeamidinated derivative, by itself or in association with the native molecule, to further deamidination. The deamidination in the initial reaction period (10 hours) did not result in any inactivation of RNase A. Chromatographic analysis of the initial reaction products on CM?cellulose showed that with the progress of the reaction, there is a gradual disappearance of RNase A from the reaction mixture. During this period, the number of moles of RNase A reacted was found to be equal to the number of moles of ammonia released. A derivative of RNase A, having the same specific activity, has been isolated from a 10?hour HCl?reaction product by chromatography on Amberlite XE?64. This derivative, designated RNase Aa?, has been found to be chromatographically distinct from RNase A. Other derivatives of RNase A having reduced specific activity have been obtained from products at later stages of reaction by CM?cellulose chromatography. It was observed that RNase Aa? is formed from RNase A by reaction in H?SO? as well. The rate of formation of this derivative in H?SO? was found to be the same as for an equivalent normality of HCl. RNase Aa? was found to be as active as RNase A both towards RNA and towards cytidine cyclic phosphate. The Km and the pH?optimum of activity of RNase Aa? were found to be the same as that of RNase A. Ultraviolet absorption spectrum of RNase Aa? was very nearly the same as that of RNase A; the environment of the tyrosine residues was apparently unaltered. The amino acid composition of the acid hydrolysate of RNase Aa? was found to be the same as that of RNase A. Moving boundary electrophoretic experiments showed that RNase Aa? is electrophoretically homogeneous and differs from RNase A in possessing one extra negative charge. Hence, it was clear that RNase Aa? is a monodeamidinated derivative of RNase A. It may be added here that RNase B (31–53) has some of the characteristic features of RNase Aa?, namely, its chromatographic behaviour on Amberlite column and electrophoretic mobility. However, the structural difference between RNase Aa? and RNase B was readily apparent from the observed lysine N?terminus of RNase Aa?. From a comparative analysis of the tryptic peptides of performic acid oxidised RNase A and RNase Aa?, the location of the deamidinated residue in RNase Aa? was tentatively identified to be in the region of residues 67 to 85 in RNase A sequence. RNase Aa? was found to be acted on by subtilisin to form active RNase S?type derivative. However, unlike the subtilisin digestion of RNase A (which proceeds to nearly 100% conversion to RNase S), the digestion of RNase Aa? was found to proceed only up to 50% conversion. The subtilisin?modified derivative of RNase Aa? (designated RNase Aa?S) was isolated by chromatography on Amberlite XE?64; this derivative was found to be chromatographically distinct from RNase S. RNase Aa?S could be fractionated by trichloroacetic acid to yield RNase Aa?S?protein and RNase Aa?S?peptide. Activity measurements showed that these components are inactive when assayed individually, but reconstitute the original activity in presence of each other. However, it was apparent from the reconstitution studies that the interaction between the protein and the peptide components of RNase Aa?S is not as strong as that between RNase S?protein and RNase S?peptide. RNase Aa?S?peptide was found to be identical with RNase S?peptide. Further, the reconstituted product formed by mixing RNase Aa?S?peptide with RNase S?protein was found to be chromatographically identical with RNase S. Similarly, the reconstituted product formed from RNase Aa?S?protein and RNase S?peptide was chromatographically identical with RNase Aa?S. RNase S was also found to undergo structural and activity changes in acidic solutions, similar to those of RNase A, and the initial reaction product was found to contain RNase Aa?S. However, unlike the HCl?treatment of RNase A, wherein the modification is only in the protein region of the molecule, the HCl?treatment of RNase S was found to affect both the S?peptide and the S?protein regions of the molecule. In RNase S, there appeared to be a slow simultaneous deamidination in the S?peptide region indicating the influence of the 20–21 peptide bond cleavage on the susceptibility of the S?peptide region for reactions in acidic solution. The modified S?peptide was, however, capable of binding to both RNase Aa?S? protein and to RNase S?protein. In conclusion, this thesis has dealt with the studies on the structural aspects of protein catalysis related to the acid denaturation of RNase A and RNase S ('conjugated?type' derivative of RNase A). The initial stable intermediate formed from RNase A is a monodeamidinated derivative (RNase Aa?) having the same specific activity as the native enzyme. The deamidinated derivative is acted upon by subtilisin at the same peptide bond as in RNase A indicating the structural similarity between RNase A and RNase Aa?. Although there are noticeable differences in the interactions between the constituent fragments of the subtilisin derivatives of RNase A and RNase Aa?, the catalytic activity of the reconstituted enzyme is the same as RNase A in both cases. In the acid denaturation of RNase A and RNase S that has been studied, stable, irreversibly chemically altered forms of the native proteins could be detected and isolated. In spite of the chemical changes in acidic solution, these proteins, simple as well as 'conjugated', have been found to be capable of regaining structures which exhibit the same catalytic activity as the native enzyme.