Gennady I. Yakovlev

The effect of net charge on the solubility, activity, and stability of ribonuclease Sa

The net charge and isoelectric pH (pI) of a protein depend on the content of ionizable groups and their pK values. Ribonuclease Sa (RNase Sa) is an acidic protein with a pI = 3.5 that contains no Lys residues. By replacing Asp and Glu residues on the surface of RNase Sa with Lys residues, we have created a 3K variant (D1K, D17K, E41K) with a pI = 6.4 and a 5K variant (3K + D25K, E74K) with a pI = 10.2. We show that pI values estimated using pK values based on model compound data can be in error by >1 pH unit, and suggest how the estimation can be improved. For RNase Sa and the 3K and 5K variants, the solubility, activity, and stability have been measured as a function of pH. We find that the pH of minimum solubility varies with the pI of the protein, but that the pH of maximum activity and the pH of maximum stability do not.

High sequence similarity between a ribonuclease from ginseng calluses and fungus-elicited proteins from parsley indicates that intracellular pathogenesis-related proteins are ribonucleases

A ribonuclease from a callus cell culture of Panax ginseng C.A. Mey strain R1 was isolated. A pure protein with an apparent molecular mass of 18 kDa was obtained. The N-terminal sequences of the protein and of the C-terminal CNBr peptide were determined. No homology with other ribonucleases was found, but there was 60–70% sequence identity with two intracellular pathogenesis-related (IPR) proteins from parsley, indicating that not only these two proteins, but also homologous IPR proteins identified in other plant species are ribonucleases.

MUTATIONAL ANALYSIS OF THE ACTIVE SITE OF RNASE OF BACILLUS INTERMEDIUS (BINASE)

To elucidate the functional role of some residues in the active site of binase, the extracellular ribonuclease of Bacillus intermedius, we used site‐directed mutagenesis. On cleavage of various substrates the catalytic activity of binase mutant His101Glu is 2.0–2.7% of that for native enzyme. The decrease in activity is determined mainly by the decrease in molecular rate constant k cat with almost unchanged affinity of the enzyme for the substrate, characterized by K M. This is the expected result if His101 acts as an general acid, donating a proton to the leaving group on cleavage of a phosphodiester bond. The replacement of Lys26 by Ala causes a reduction in the enzyme activity to 13—33%, depending on the substrate. The activity decreases are due to changes in both k cat and K M for poly(I) and poly(A) but in k cat alone for GpA. In the latter case the effect is far less than that seen in the homologous mutation in the closely related enzyme, barnase.

Synthesis and properties of 3′-C-methylnucleosides and their phosphoric esters

Abstract 3′- C -Methyluridine and 3′- C -methylcytidine were synthesized in 11 steps starting from d -glucose. The conformation of 3′- C -methylnucleosides was studied in solution and in the crystal by using the techniques of c.d., 1 H-n.m.r. spectroscopy, and X-ray diffraction analysis. 3′- C -Methyluridine 2′,3′-cyclophosphate was prepared, and its hydrolysis with nucleases was studied. 3′- C -Methyluridine 5′- mono- and 5′-tri-phosphate were also synthesized.

Contribution of active site residues to the activity and thermal stability of ribonuclease Sa

We have used site‐specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000‐fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, kcat is reduced sixfold despite the fact that Glu 74 is over 15 Å from the active site. The pH dependence of kcat/KM is very similar for Glu74Lys and wild‐type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild‐type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability.

Thermodynamics of denaturation of complexes of barnase and binase with barstar.

Differential scanning calorimetry was used to study the thermodynamics of denaturation of protein complexes for which the free energy stabilizing the complexes varied between -8 and -16 kcal/mol. The proteins studied were the ribonucleases barnase and binase, their inhibitor barstar and mutants thereof, and complexes between the two. The results are in good agreement with the model developed by Brandts and Lin for studying the thermodynamics of denaturation for tight complexes between two proteins which undergo two-state thermal unfolding transitions.

Dissociation constants and thermal stability of complexes of Bacillus intermedius RNase and the protein inhibitor of Bacillus amyloliquefaciens RNase

Binase, the extracellular ribonuclease of Bacillus intermedius, is inhibited by barstar, the natural protein inhibitor of the homologous RNase, barnase, of B. intermedius. The dissociation constants of the binase complexes with barstar and its double Cys40.82 Ala mutant are about 10−12 M, only 5 to 43 times higher than those of the barnase‐barstar complex. As with barnase, the denaturation temperature of binase is raised dramatically in the complex. Calorimetric studies of the formation and stability of the binase‐barstar complex show that the binase reaction with barstar is qualitatively similar to that of barnase but some significant quantitative differences are reported.

Thermostability of the barnase—barstar complex

Scanning microcalorimetry was used to study heat denaturation of barnase in complex with its intracellular inhibitor barstar. The heat denaturation of the barnase—barstar complex is well approximated by two two‐state transitions with the lower temperature transition corresponding to barstar denaturation and the higher temperature one to barnase denaturation. The temperature of barnase melting in its complex with barstar is 20°C higher than that of the free enzyme. The barstar melting temperature is almost the same in the complex or alone (71°C at pH 6.2 and 68°C at pH 8.0). It seems possible that when barstar unfolds it can remain bound to barnase, while the latter unfolds only on dissociation of the denatured barstar.

Proton magnetic resonance studies of DES‐(121–124)‐ribonuclease A

Bovine pancreatic ribonuclease A (RNAase A) has been extensively investigated from the point of view of structure and function [l] . NMR spectroscopy has been successful in providing information about the state of RNAase A in aqueous solution, estimates of the pK values for each histidine residue and an evaluation of the enzyme-inhibitor complex structure 12-51. In this paper we report the PMR data for des-( 121124, Asp-Ala-Ser-Val-)-RNAase A, which was obtained by a limited hydrolysis of RNAase A and was designated as PIR [6-81. The data presented here on the pH-dependence of PMR spectra of PIR and its complexes with cytidine3’-phosphate (3’CMP) were used to estimate the individual pK’s (apparent) for the four histidines in PIR as well as for a comparison of the conformations of both proteins. The experimental data thus obtained suggest the role of Asp-121 in maintaining the precise orientation of the essential functional groups in the active site of RNAase A.

Determination of the nucleotide conformation in the productive enzyme-substrate complexes of RNA-depolymerases

© 1997 Federation of European Biochemical Societies.