Ribonuclease substrate specificity

A very different ribonuclease participates in the biosynthesis of all of the transfer RNAs of E. coli. Ribonuclease P cuts a 5 leader sequence from precursor RNAs to form the final 5 termini of the tRNAs. Sidney Altman and coworkers in 1980 showed that the enzyme consists of a 13.7-kDa protein together with a specific 377-nucleotide RNA component (designated Ml RNA) that is about five times more massive than the protein.779 Amazingly, the Ml RNA alone is able to catalyze the ribonuclease reaction with the proper substrate specificity.780 7823 The protein apparently accelerates the reaction only about twofold for some substrates but much more for certain natural substrates. The catalytic center is in the RNA, which functions well only in a high salt concentration. A major role of the small protein subunit may be to provide counterions to screen the negative charges on the RNA and permit rapid binding of substrate and release of products.783 Eukaryotes, as well as other prokaryotes, have enzymes similar to the E. coli RNase R However, the eukaryotic enzymes require the protein part as well as the RNA for activity.784... 

Enzymes which catalyze the hydrolysis of the unit linkage of sequential residues of oligomers or polymers determine their substrate specificity by recognizing the particular unit residue in the sequential chain as well as the direction of the chain. For example, ribonuclease cleaves the 3 -phosphate of a pyrimidine nucleotide residue but not the 5 -phosphate, and trypsin hydrolyzes peptide bonds which involve the arginine or lysine residue at the carbonyl end but not at the amino end. This is also the case for the hydrolysis of a variety of synthetic substrates and quasi-substrates (Sect. 4.1). Synthetic trypsin substrates are ester or amide derivatives in which the site-specific group (positive charge) is contained in their carbonyl portion. 

A series of carboxyl derivatized polyglucoses were studied as inhibitors of ribonuclease activity, in an attempt to relate charge density to inhibitory activity.202 In comparison with other factors, it was concluded that coulombic forces probably play a major role in complex-formation between enzyme and substrate, and between enzyme and inhibitor. However, other specific, nonelectrostatic forces were shown to participate in the binding of bovine pancreatic ribonuclease to ribonucleic acid.204... 

Bovine pancreatic ribonuclease catalyzes the hydrolysis of RNA by a two-step process in which a cyclic phosphate intermediate is formed (equation 16.35). The cyclization step is usually much faster than the subsequent hydrolysis, so the intermediate may be readily isolated. DNA is not hydrolyzed, as it lacks the 2 -hydroxyl group that is essential for this reaction. There is a strong specificity for the base B on the 3 side of the substrate to be a pyrimidine—uracil or cytosine. 

Three levels of protein-nucleic acid recognition have been observed. Nature provides three examples of protein-nucleic acid interactions which we shall consider. The nucleic acid component can be (1) a single nucleotide, e.g., a coenzyme or a substrate, (2) a single-stranded DNA or RNA as in ribonucleases A and T, or (3) a double-stranded DNA or RNA as in the highly specific complexes with repressors in the tRNA-synthetase complex, or in the unspecific nuclease DNase I. 

But an important point is to realize that (o) comparisons at the molecular level cannot be established between species, since the same enzyme formed by various species may have different specific activities and require different cofactors. (6) Very large errors can be made, and actually have been made in the past, by using poor techniques for the determination of enzyme activity. At the present time, directly active enzymes such as amylase (19, 20), lipase (16), and ribonuclease (21) are measured with a reasonable degree of accuracy. Good techniques and specific substrates are also available for chymotrypsin, trypsin, carbox3 ptidases A and B. But the special problem here is that proteolytic enzymes must be activated and that the activation step must be carefully controlled in complex mixtures (Figs. 5 and 6). 

Table VII summarizes the conditions for chymotryptic hydrolysis of the proteins and peptides listed in Table VI. The parameters which would be expected to determine the rate of hydrolysis (apart from the nature of the bonds in the particular substrates) are temperature, pH, time of hydrolysis, and the molar ratio of chymotrypsin to substrate. All these factors often differ considerably for the substrates listed. Hydrolyses have been performed under conditions which vary from 2 to 24 hr, from pH 7.0 to 9.0, from 22° to 40°C, and at enzyme to substrate molar ratios between 1/360 to 1/21. It is not obvious how variations in pH and temperature affect the apparent specificity of chymotrypsin, but at low molar ratios of enzyme to substrate only the most susceptible bonds would be expected to be hydrolyzed. The lowest molar ratio was employed in the studies with ribonuclease. The only bonds of an unusual nature which were split were those formed by serine and histidine in the following sequences, -Thr-Ser. . . Ala-Ala- and -Lys-His. . . Ileu-Ileu-. Many of the unusual splits listed in Table VI were observed in equine or human cytochrome c and in oxidized papain. Each of these substrates was digested for long periods of time and at high ratios of enzyme to substrate under conditions which would be expected to split bonds that are usually resistant to hydrolysis.

A number of studies have been designed to determine the nature of the mechanism of proteolysis with a specific enzyme and substrate. Ginsberg and Schachman (1960a,b) concluded that chymotryptic hydrolysis of insulin probably proceeds by the all or none mechanism, whereas peptic hydrolysis of ribonuclease follows a zipper mechanism. 

This final acid phosphatase preparation had a specific activity of 468 and represented an approximately 1900-fold purification of the acid phosphatase in the starting crude spleen nuclease II. It contained no acid deoxyribonuclease, acid ribonuclease, exonuclease, and phosphodiesterase activities that could be detected in a 0.1-ml sample after 2 hours of incubation with the appropriate substrate. The relative rates of hydrolysis of various substrates were as follows p-nitrophenyl phosphate, 100 5 -AMP, 63 j8-glycerophosphate, 60 ATP, 0. With p-nitrophenyl phosphate as substrate, the pH optimum was broad and lay between pH 3.0 and pH 4.8. The Michaelis constant at 37°C was 7.25 X 10" mM. Phosphate and chloride ions acted as competitive inhibitors. 

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