Roles of Lysine1 and Lysine7 Residues of Bovine Pancreatic Ribonuclease in the Enzymatic Activity

Irie, Masachika; Ohgi, Kazuko; Yoshinaga, Makiko; Yanagida, Tamami; Okada, Yoshio; Teno, Naoki

doi:10.1093/oxfordjournals.jbchem.a121785

Cited by 12 publications

(3 citation statements)

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“…Several previous reports support this role for Lys-7. Thus experiments with oligomeric substrates and an RNAase S' with a modified Speptide ([7-norleucine]S-peptide) clearly support the involvement of Lys-7 in the interaction with the phosphate group that binds in P2 (Irie et al, 1986). Detailed X-ray-crystallographic studies of a complex between RNAase A and d(pA)4 indicate that the phosphate adjacent to that occupying p1, i.e.…”

mentioning

confidence: 78%

Chemical modification by pyridoxal 5′-phosphate and cyclohexane-1,2-dione indicates that Lys-7 and Arg-10 are involved in the p2 phosphate-binding subsite of bovine pancreatic ribonuclease A

Richardson

Parés

Cuchillo

1990

Biochemical Journal

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Steric and chemical evidence had previously shown that residues Lys-7 and/or Arg-10 of bovine pancreatic RNAase A could belong to the p2 phosphate-binding subsite, adjacent to the 3' side of the main site p1. In the present work chemical modification of the enzyme with pyridoxal 5'-phosphate and cyclohexane-1,2-dione was carried out in order to identify these residues positively as part of the p2 site. The reaction with pyridoxal 5'-phosphate yields three monosubstituted derivatives, at Lys-1, Lys-7 and Lys-41. A strong decrease in the yield of derivatives at Lys-7 and Lys-41 was observed when either p1 or p2 was specifically blocked by 5'-AMP or 3'-AMP respectively. These experiments indicate that both sites are needed for the reaction of pyridoxal 5'-phosphate with RNAase A to take place. The positive charge in one of the sites interacts with the phosphate group of pyridoxal 5'-phosphate, giving the proper orientation to the carbonyl group, which then reacts with the lysine residue present in the other site. The absence of reaction between pyridoxal 5'-phosphate and an RNAase derivative that has the p2 site blocked supports this hypothesis. Labelling of Lys-7 with pyridoxal 5'-phosphate has a more pronounced effect on the kinetics with RNA than with the smaller substrate 2',3'-cyclic CMP. In addition, when the phosphate moiety of the 5'-phosphopyridoxyl group was removed with alkaline phosphatase the kinetic constants with 2',3'-cyclic CMP returned to values very similar to those of the native enzyme, whereas a higher Km and lower Vmax. were still observed for RNA. This indicates that this new derivative has recovered a free p1 site and, hence, the capability to act on 2',3'-cyclic CMP, but the presence of the pyridoxyl group bound to Lys-7 is still blocking a secondary phosphate-binding site, namely p2. Finally, reaction of cyclohexane-1,2-dione at Arg-10 is suppressed in the presence of 3'-AMP but only a 19% decrease is observed with 5'-AMP, suggesting that Arg-10 is also close to the p2 phosphate-binding subsite.

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confidence: 78%

Chemical modification by pyridoxal 5′-phosphate and cyclohexane-1,2-dione indicates that Lys-7 and Arg-10 are involved in the p2 phosphate-binding subsite of bovine pancreatic ribonuclease A

Richardson

Parés

Cuchillo

1990

Biochemical Journal

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“…It seems unlikely that this increase resulted from the use of deoxyadenosine, since no difference in the kinetic parameters for UpA and UpdA with RNase A have been found (2). Previously, such improved catalytic efficiency with elongated substrates has been explained by the interaction between the additional phosphate groups and positively charged subsites on the enzyme (44,45). In this model is bound by Lys-66, whereas the one on the 3'-side of the purine (the p2-subsite) is bound by Lys-7 and Arg-10.…”

Section: Discussionmentioning

confidence: 99%

A novel fluorogenic substrate for ribonucleases. Synthesis and enzymatic characterization

Zelenko¹,

Neumann²,

Brill

et al. 1994

Nucl Acids Res

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The synthesis and enzymatic characterization of DUPAAA, a novel fluorogenic substrate for RNases of the pancreatic type is described. It consists of the dinucleotide uridylyl-3',5'-deoxyadenosine to which a fluorophore, o-aminobenzoic acid, and a quencher, 2,4-dinitroaniline, have been attached by means of phosphodiester linkages. Due to intramolecular quenching the intact substrate displayed very little fluorescence. Cleavage of the phosphodiester bond at the 3'-side of the uridylyl residue by RNase caused a 60-fold increase in fluorescence. This allowed the continuous and highly sensitive monitoring of enzyme activity. The substrate was turned over efficiently by RNases of the pancreatic type, but no cleavage was observed with the microbial RNase T1. Compared to the dinucleotide substrate UpA, the specificity constant with RNase A, RNase PL3 and RNase U(s) increased 6-, 18-, and 29-fold, respectively. These differences in increased catalytic efficiency most likely reflect differences in the importance of subsites on the enzyme in the binding of elongated substrates. Studies on the interactions of RNase inhibitor with RNase A using DUPAAA as a reporter substrate showed that it was well suited for monitoring this very tight protein-protein interaction using pre-steady-state kinetic methods.

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“…The reaction yielded a major derivative (derivative II) with the nucleotide label attached to the ␣-amino group. Different studies suggested that the phosphate group was bound in a specific phosphate-binding subsite, p 2 , and that Lys-7 and Arg-10 were involved in that subsite (Arú s et al, 1981;Irie et al, 1986;de Llorens et al, 1989; Richardson et al, 1990). The structure of derivative II was recently solved by x-ray crystallography (Boqué et al, 1994), and the structure found is in accordance with the location of p 2 , B 3 , and R 3 2 subsites at the N-terminal region of the protein.…”

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confidence: 99%

The Role of Non-catalytic Binding Subsites in the Endonuclease Activity of Bovine Pancreatic Ribonuclease A

Moussaoui

Guasch

Boix

et al. 1996

Journal of Biological Chemistry

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Bovine pancreatic ribonuclease A catalyzes the depolymerization of RNA. There is much evidence that several subsites, in addition to the main catalytic site, are involved in the formation of the enzyme-substrate complex. This work analyzes the pattern of oligonucleotide formation by ribonuclease A using poly(C) as substrate. The poly(C) cleavage shows that the enzyme does not act in a random fashion but rather prefers the binding and cleavage of the longer substrate molecules and that the phosphodiester bond broken should be 6 -7 residues apart from the end of the chain to be preferentially cleaved by ribonuclease A. The results demonstrate the model of the cleavage of an RNA chain based on the cooperative binding between the multisubsite binding structure of ribonuclease A and the phosphates of the polynucleotide (Paré s, X., Nogué s, M. V., de Llorens, R., and Cuchillo, C. M. (1991) in Essays in Biochemistry (Tipton, K. F., ed) Vol. 26, pp. 89 -103, Portland Press Ltd., London). The contribution to the enzymatic process of the non-catalytic phosphate-binding subsite (p 2 ) adjacent to the catalytic center has been analyzed in p 2 chemically modified ribonuclease A or by means of sitedirected mutagenesis. In both cases deletion of p 2 abolishes the endonuclease activity of ribonuclease A, which is substituted by an exonuclease activity. All these results support the role of the multisubsite structure of the enzyme in the endonuclease activity and in the catalytic mechanism.Bovine pancreatic ribonuclease A (RNase A) 1 (EC 3.1.27.5) is a well known enzyme whose main physical, chemical, and enzymatic properties have been the subject of extensive reviews (Richards and Wyckoff, 1971;Blackburn and Moore, 1982;Eftink and Biltonen, 1987). RNase A is an endonuclease that cleaves 3Ј,5Ј-phosphodiester linkages of single-stranded RNA when the base of the nucleotide in the 3Ј position is a pyrimidine. The use of well defined low molecular mass substrates such as pyrimidine 2Ј,3Ј-cyclic mononucleotides and dinucleoside monophosphates provided most of the kinetic data on which all mechanistic studies on RNase A have relied. Only a few kinetic studies have been carried out with longer oligonucleotides and homopolynucleotides such as poly(U) (Irie et al., 1984a(Irie et al., , 1984b, but no detailed studies with RNA as substrate have been carried out. The reasons for this are (i) the difficulties in the kinetic analysis derived from the complex structural features of the RNA molecule, (ii) the difficulty of monitoring a very fast reaction in a reliable fashion, and (iii) the spectrophotometric methods used can only give an average measure of all the species produced during the reaction, without giving any idea about either the size distribution of the products or the characteristics of the bond broken. The use of HPLC techniques has circumvented some of the problems concerning the analysis of the products of the reaction. Using an anion exchange column it was possible to measure the products of the reaction at high concentration...

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Roles of Lysine1 and Lysine7 Residues of Bovine Pancreatic Ribonuclease in the Enzymatic Activity

Cited by 12 publications

References 0 publications

Chemical modification by pyridoxal 5′-phosphate and cyclohexane-1,2-dione indicates that Lys-7 and Arg-10 are involved in the p2 phosphate-binding subsite of bovine pancreatic ribonuclease A

Chemical modification by pyridoxal 5′-phosphate and cyclohexane-1,2-dione indicates that Lys-7 and Arg-10 are involved in the p2 phosphate-binding subsite of bovine pancreatic ribonuclease A

A novel fluorogenic substrate for ribonucleases. Synthesis and enzymatic characterization

The Role of Non-catalytic Binding Subsites in the Endonuclease Activity of Bovine Pancreatic Ribonuclease A

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