Proton nuclear magnetic resonance studies of histidine residues in rat and other rodent pancreatic ribonucleases. Effects of pH and inhibitors

Proton nuclear magnetic resonance spectra of the histidine residues in bovine and rat ribonuclease have been compared. The changes in chemical shift on titration and on binding of cytidine-3′-monophosphate and cytidine-2′-monophosphate have been followed. In the presence of the cytidine derivatives the spectra of both enzymes resemble each other more than those of the free enzymes. With these inhibitors, two histidines in rat ribonuclease exhibit the same pK values and shifts as the active site residues histidine 12 and 119 in the bovine enzyme. Their pK values in the inhibitor-free rat enzyme are about 0.4 higher than in the beef enzyme, which can be explained by the substitution at the entrance of the active site cleft of arginine 39 in the beef enzyme by serine in the rat enzyme. Rat ribonuclease contains one histidine with a rather high pK value of 7.6. The cytidine derivatives affect its chemical shift in exactly the same way as the shift of histidine 48 in bovine ribonuclease. The high pK value of this residue in rat ribonuclease can be explained by assuming a strong hydrogen bridge with glutamic acid 16. The other two histidines in rat ribonuclease have rather low pK values of 6.1 and 6.3. The histidine with a pK value of 6.3 has been assigned to position 105 and that with a pK value of 6.1 to position 73.The closer resemblance of the active sites of bovine and rat ribonuclease in the presence of inhibitors than in the inhibitor-free enzymes makes the concept of induced fit interesting from an evolutionary point of view.The characteristic downfield shift of the protonated form of histidine 119 in the complexes of bovine and rat ribonuclease with cytidine-3′-monophosphate is not observed with uridine-3′-monophosphate, suggesting non-identical binding of these pyrimidine nucleotides.Some preliminary results on the nuclear magnetic resonance properties of the histidine residues in coypu and chinchilla pancreatic ribonuclease have been obtained.     

Evolution of nucleic acids coding for ribonucleases: the mRNA sequence of mouse pancreatic ribonuclease

The cDNA of mouse pancreatic mRNA has been cloned. After the library was screened with a rat ribonuclease cDNA probe, the positive clones were isolated and sequenced. There were no differences from the previously determined protein sequence. The mRNA codes for a preribonuclease of 149 amino acid residues including a signal peptide of 25 amino acids. The 3' noncoding region has a length of 260 bp, and the total mRNA length is approximately 940 bp. Comparison with the rat pancreatic ribonuclease sequence showed a high rate of nucleotide substitution. Within the coding region, nonsynonymous and synonymous substitution rates are 4.3 X 10(-9) and 15 X 10(-9) nucleotide substitutions/site/year, respectively. The latter value is one of the highest rates observed in the molecular evolution of mammalian nuclear genes. In the signal sequences the synonymous substitution rate is much lower and about the same as the nonsynonymous rate. Signal sequences of other mouse and rat proteins also exhibit little difference between synonymous and nonsynonymous rates. The sequences of rat and mouse pancreatic ribonuclease messengers were compared with those of bovine pancreatic, seminal, and brain ribonuclease. While the 3' noncoding regions of rat and mouse are very similar, as are those of the three bovine messengers, there is no significant similarity between both rodent and the three bovine messengers for the greater part of these regions. There is a duplication of approximately 50 nucleotides in the 3' noncoding region of the bovine messengers, with a region rich in A and C in between. The presence of this structural feature may be correlated with recent gene duplications that have occurred in the bovine genome.     

Kinetic studies on turtle pancreatic ribonuclease: a comparative study of the base specificities of the B2 and P0 sites of bovine pancreatic ribonuclease A and turtle pancreatic ribonuclease

Kinetic constants for the transesterification of eight dinucleoside phosphates CpX and UpX by bovine and turtle pancreatic ribonuclease were determined. Both ribonucleases have a preference for purine nucleotides at the position X. However, bovine ribonuclease, like other mammalian ribonucleases, prefers 6-amino bases at this site, while turtle ribonuclease prefers 6-keto bases. This difference in specificity at the B2 site may be explained by the substitution of glutamic acid at position 111 by valine in turtle ribonuclease. These results have been confirmed by inhibition studies with the four nucleoside triphosphates. Inhibition studies with pT and pTp showed that a cationic binding group (P0) for the 5'-phosphate of the pyrimidine nucleotides bound at the primary B1 site is present in turtle ribonuclease, although lysine at position 66 in bovine ribonuclease is absent in turtle ribonuclease. However, the side chain of lysine 122 in turtle ribonuclease is probably located in the correct position to take over the role as cationic P0 site.     

The nature of the circular-dichoric spectra of complexes between ribonuclease A and nucleotides.

The circular-dichroism and proton-magnetic-resonance spectra of complexes of ribonuclease A with dihydrouridine 3'-phosphate, 2'- and 3'-CMP, arabinosyl-3'-CMP, 1-(2-hydroxyethyl)cytosine 2'-phosphate and 1-(3-hydroxypropyl)cytosine 3'-phosphate were studied. Comparison of the results shows that non-additivity of the circular-dichroic spectrum of an enzyme-nucleotide complex may be due to: (a), alteration of the circular dichroic spectrum of the nucleotide under the influence of the asymmetric protein matrix (induced dichroism), and (b) a change in the nucleotide conformation. The contribution of each of the two factors was estimated to calculate the circular-dichoroic spectra of 2'-CMP and 3'-CMP in complex with ribonuclease A. 3'-CMP in this complex was characterized by negative circular dichroism in the long-wavelength absorption band of the nucleotide, whereas 2'-CMP was characterized by positive circular dichroism. Since both nucleotides in the complex are known to be in an anti conformation, it follows that even small changes in the conformation considerably modify the circular-dichroic spectrum of the nucleotide in complex with the enzyme.     

The unswapped chain of bovine seminal ribonuclease: Crystal structure of the free and liganded monomeric derivative

Bovine seminal ribonuclease, a homodimeric enzyme joined covalently by two interchain disulphide bonds, is an equilibrium mixture of two conformational isomers, MxM and M=M. The major form, MxM, whose crystal structure has been previously determined at 1.9 A resolution, presents the swapping of the N-terminal segments (residues 1-15) and composite active sites formed by residues of different chains. The three-dimensional domain swapping does not occur in the M=M form. The different fold of each N-terminal tail is directed by the hinge loop (residue 16-22) connecting the swapping domain to the body of the protein. Reduction and alkylation of interchain disulphide bridges produce a monomeric derivative and a noncovalent swapped dimer, which are both active. The free and nucleotide-bound forms of the monomer have been crystallized at an alkaline pH and refined at 1.45 and 1.65 A resolution, respectively. In both cases, the N-terminal fragment is folded on the main body of the protein to produce an intact active site and a chain architecture very similar to that of bovine pancreatic ribonuclease. In this new fold of the seminal chain, the hinge loop is disordered. Despite the difference between the tertiary structure of the monomer and that of the chains in the MxM form, the active sites of the two enzymes are virtually indistinguishable. Furthermore, the structure of the liganded enzyme represents the first example of a ribonuclease complex studied at an alkaline pH and provides new information on the binding of a nucleotide when the catalytic histidines are deprotonated.     

Topological comparison of two helix destabilizing proteins: ribonuclease A and the gene 5 DNA binding protein

A topological comparison of the two helix destabilizing proteins, pancreatic ribonuclease A and the gene 5 DNA binding protein of bacteriophage fd has been completed utilizing the available high resolution tertiary structures of each protein. The results indicate these two proteins are structurally if not also evolutionarily related. Regions of closet topological equivalence occur between beta loops directly involved in nucleotide binding or are required for the maintenance of their respective oligonucleotide binding channels. In addition, there is a similar placement of critical amino acid side chains about the binding site. Further evidence for this structural relationship is obtained by comparison of structural data for the mode of complexation of polynucleotides to each protein. The results of topological comparison suggest the essential property shared by helix destabilizing proteins, whether specialized DNA binding proteins such as G5BP or proteins with other primary functional roles, like ribonuclease A, is the presence of an elongated oligonucleotide binding channel. Although ribonuclease A and G5BP are structurally related, it seems likely any protein with this structural feature will exhibit a helix destabilizing capacity. This conclusion is supported by the diversity of molecular characteristics shown by other proteins having this activity.     

Computer modeling studies on the subsite interactions of ribonuclease T1.

The modes of binding of pGp,ApG,CpG and UpG to the enzyme ribonuclease T1 were determined by computer modeling. Essentially two binding modes are possible for all the four ligands--one with the 3'-phosphate group occupying the phosphate binding site (substrate mode of binding) and the second with the 5'-phosphate group occupying the phosphate binding site (inhibitor mode of binding). The latter binding mode is energetically favoured over the former and in this mode the base (G) and the 5'-phosphate moieties occupy the same sites on the enzyme as 5'-GMP when bound to RNase T1. The ribose moiety of pGp adopts a C3'-endo pucker form when bound to the enzyme and the glycosyl torsion angle will be in -syn range as 5'-GMP in the RNase T1-5'-GMP complex. Based on these results, a mechanism for the release of the product subsequent to cleavage of the substrate by the enzyme has been proposed. The amino acid residues Asn98 and Tyr45 are shown to form the subsites for the phosphate and the base respectively on the 5'-side of the guanine occupying the primary binding site. These studies also provide a stereochemical explanation for the specificity of the 1N subsite for adenine.     

Nucleotide binding and affinity labelling support the existence of the phosphate-binding subsite p2 in bovine pancreatic ribonuclease A

When the reaction of bovine pancreatic ribonuclease A with 6-chloropurine riboside 5'-monophosphate was carried out in the presence of several natural mononucleotides, a decrease of 25-75% was found in the amount of the reaction product derivative II (the main product of the reaction which has the nucleotide label at the alpha-NH2 group of Lys-1). The efficiency of inhibition followed the order 3'-AMP greater than 5'CMP approximately equal to 5'AMP greater than 3'CMP. Previous studies indicate that this order reflects the extent of occupancy of p2, a phosphate-binding subsite adjacent to the catalytic centre. This finding suggests that derivative II is the result of affinity labelling and that the phosphate group of the halogenated nucleotide binds to p2 before the reaction takes place. The dissociation constants and stoichiometry of the interaction between native enzyme, derivative II and derivative E (homologous to derivative II, but labelled with a nucleoside instead of a nucleotide) with 3'AMP and 5'AMP at several pH values were also determined. Although in general one strong binding site was found, no strong binding occurs between 3'AMP and derivative II. It is concluded that the phosphate of the label occupies the same site p2, as the phosphate of 3'AMP. Finally, the pH dependence for the binding of 3'AMP and 5'AMP to RNAase A indicates that they bind to different protein groups. The results presented support the structure of the active site of ribonuclease A postulated previously (Parés, X., Llorens, R., Arús, C. and Cuchillo, C.M. (1980) Eur. J. Biochem. 105, 571-579).     

Isolation and characterization of a human colon carcinoma-secreted enzyme with pancreatic ribonuclease-like activity

A ribonuclease was isolated from serum-free supernatants of the human colon adenocarcinoma cell line HT-29. It was purified by cation-exchange and C18 reversed-phase high-performance liquid chromatography. The protein is basic, has a molecular weight of approximately 16,000, and has an amino acid composition that is significantly different from that of human pancreatic ribonuclease. The amino terminus is blocked, and the carboxyl-terminal residue is glycine. The catalytic properties of this ribonuclease resemble those of the pancreatic ribonucleases in numerous respects. Thus, it exhibits a pH optimum of approximately 6 for dinucleotide cleavage and employs a two-step mechanism in which transphosphorylation to a cyclic 2',3'-phosphate is followed by slower hydrolysis to produce a 3'-phosphate. It does not cleave NpN' substrates in which adenosine or guanosine is at the N position and prefers purines at the N' position. Like bovine ribonuclease A, the HT-29-derived ribonuclease is inactivated by reductive methylation or by treatment with iodoacetate at pH 5.5 and is strongly inhibited by the human placental ribonuclease inhibitor. However, in contrast, the tumor enzyme does not cleave CpN bonds at an appreciable rate and prefers poly(uridylic acid) as substrate 1000-fold over poly(cytidylic acid). It also hydrolyzes cytidine cyclic 2',3'-phosphate at least 100 times more slowly than uridine cyclic 2',3'-phosphate and is inhibited much less strongly by cytidine 2'-monophosphate than by uridine 2'-monophosphate. Other ribonucleases known to prefer poly(uridylic acid) were isolated both from human serum and from liver and were compared with the tumor enzyme. The physical, functional, and chromatographic properties of the serum ribonuclease are essentially identical with those of the tumor enzyme. The liver enzymes, however, differ markedly from the HT-29 ribonuclease. The potential utility of the tumor ribonuclease in the diagnosis of cancer is considered.     

Photochemical labeling of bovine pancreatic ribonuclease A with 8-azidoadenosine 3',5'-bisphosphate

A simple method has been developed for the preparation of 5'-32P-labeled 8-azidoadenosine 3',5'-bisphosphate (p8N3Ap) for use in photoaffinity labeling studies. Irradiation of a complex between p8N3Ap and bovine pancreatic ribonuclease A (RNase A) with light of 300-350 nm led to the covalent attachment of the nucleotide to the enzyme. RNase A could also be labeled in the dark with prephotolyzed p8N3Ap. In either case, the nucleotide reacted with the same tryptic peptide, encompassing amino acids 67-85 of the protein. The site of labeling was determined to be either Thr-78 or Thr-82, both of which are close to or at the pyrimidine binding site of the enzyme. This result is consistent with recent nuclear magnetic resonance and X-ray studies which indicate that 8-substituted adenine nucleotides interact with the pyrimidine binding site of RNase A.     

Substrate specificity of acetyl-CoA-carboxylase from the rat liver

The interaction between acetyl-CoA fragments and rat liver acetyl-CoA carboxylase was studied. It was found that the 3'-phosphate group did not interfere with the enzyme interaction since the substrate properties of acetyl-dephospho-CoA and acetyl-CoA are nearly identical. The non-nucleotide substrate analogs S-acetyl-pantethin and its 4'-phosphate) also displayed substrate properties (V = 1.5% and 15% of the V for acetyl-CoA carboxylation respectively). The nucleotide fragment of the acetyl-CoA molecule produced an appreciable effect on the thermodynamics of this substrate interaction with the enzyme. Its physiological role consists in all probability, in the activation and propes orientation of the acetyl group in the enzyme active center. The far more pronounced substrate properties of S-acetyl pantethin 4'-phosphate and the inhibitory properties of pantethin 4'-phosphate (compared to non-phosphorylated analogs) suggest the essential role of the beta-phosphate residue of ADP in the acetyl-CoA binding to the enzyme. The data obtained suggest also that the hydrophobic region responsible for the acyl radical binding, has a site which specifically recognizes the beta-mercaptoethyl residue of the CoA pantethin fragment. The pivotal role in the acetyl-CoA carboxylase interaction with the substrate is ascribed to the productive binding of the acetyl radical; the contribution of individual fragment of the CoA molecule is variable.     

Acceptor activity of 4-N-acetylcytidine in the synthesis of (3'-5')-internucleotide bond catalyzed by pancreatic nuclease]

Cytidine and 4-N-acetylcytidine were compared as phosphate acceptors in dinucleoside monophosphate synthesis catalyzed by pancreatic ribonuclease with uridine-2',3'-cyclophosphate and cytidine-2',3'-cyclo phosphate as phosphate donors. Because of low solubility of 4-N-acetylcytidine in water, the synthesis was carried out in aqueus-organic media. The results obtained indicate that acetylation of the exoaminogroup of cytidine decreases its acceptor activity. For the first time uridilyl-(3'-5')-4-N-acetylcytidine and cytidilyl-(3'-5')-4-N-acetylcytidine are prepared enzymatically by pancreatic ribonuclease.     

Topological Comparison of Two Helix Destabilizing Proteins: Ribonuclease A and the Gene 5 DNA Binding Protein

Abstract

A topological comparison of the two helix destabilizing proteins, pancreatic ribonuclease A and the gene S DNA binding protein of bacteriophage fd has been completed utilizing the available high resolution tertiary structures of each protein. The results indicate these two proteins are structurally if not also evolutionarily related. Regions of closest topological equivalence occur between beta loops directly involved in nucleotide binding or are required for the maintenance of their respective oligonucleotide binding channels. In addition, there is a similar placement of critical amino acid side chains about the binding site. Further evidence for this structural relationship is obtained by comparison of structural data for the mode of complexation of polynucleotides to each protein. The results of topological comparison suggest the essential property shared by helix destabilizing proteins, whether specialized DNA binding proteins such as G5BP or proteins with other primary functional roles, like ribonuclease A, is the presence of an elongated oligonucleotide binding channel. Although ribonuclease A and G5BP are structurally related, it seems likely any protein with this structural feature will exhibit a helix destabilizing capacity. This conclusion is supported by the diversity of molecular characteristics shown by other proteins having this activity.     

Nucleotide Binding Site Communication in Arabidopsis thaliana Adenosine 5'-Phosphosulfate Kinase

Adenosine 5'-phosphosulfate kinase (APSK) catalyzes the ATP-dependent synthesis of adenosine 3'-phosphate 5'-phosphosulfate (PAPS), which is an essential metabolite for sulfur assimilation in prokaryotes and eukaryotes. Using APSK from Arabidopsis thaliana, we examine the energetics of nucleotide binary and ternary complex formation and probe active site features that coordinate the order of ligand addition. Calorimetric analysis shows that binding can occur first at either nucleotide site, but that initial interaction at the ATP/ADP site was favored and enhanced affinity for APS in the second site by 50-fold. The thermodynamics of the two possible binding models (i.e. ATP first versus APS first) differs and implies that active site structural changes guide the order of nucleotide addition. The ligand binding analysis also supports an earlier suggestion of intermolecular interactions in the dimeric APSK structure. Crystallographic, site-directed mutagenesis, and energetic analyses of oxyanion recognition by the P-loop in the ATP/ADP binding site and the role of Asp(136), which bridges the ATP/ADP and APS/PAPS binding sites, suggest how the ordered nucleotide binding sequence and structural changes are dynamically coordinated for catalysis.     

Intrachain disulfide bridges of bovine seminal ribonuclease

The pairing of the four intrachain disulfide bonds of bovine seminal ribonuclease, a dimeric protein isolated from bovine seminal plasma, has been established by the isolation and characterization of the cystine peptides obtained from a thermolytic-tryptic hydrolysate of the protein. These disulfide bonds involve eight half-cystine residues located in the protein subunit chain at sequence positions identical with those of the eight half-cystine residues of the strictly homologous chain of bovine pancreatic ribonuclease. The results reported show that these eight 'homologous' half-cystine residues pair in seminal ribonuclease exactly as they do in pancreatic ribonuclease. They also indirectly confirm that the remaining two half-cystine residues present in each chain of the seminal enzyme are involved in intersubunit bonds.     

Eosinophil cationic protein cDNA. Comparison with other toxic cationic proteins and ribonucleases

Human eosinophil granules contain several basic proteins including eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN) and major basic protein (MBP). ECP and MBP are potent helminthotoxins while EDN is less so. Both ECP and EDN possess neurotoxic and ribonuclease activities. A clone representing ECP mRNA was isolated from an eosinophil lambda ZAP cDNA library. The cDNA sequence codes for a preprotein of 160 amino acids and a protein of 133 amino acids, the amino terminus of which is identical to the known partial amino acid sequence of ECP. The ECP nucleotide sequence shows similarity to EDN, rat pancreatic ribonuclease, and human angiogenin; all are members of the ribonuclease gene superfamily. Although the deduced amino acid sequence of ECP shares identical active site and substrate binding site residues with EDN, angiogenin, and human pancreatic ribonuclease, the ribonuclease activity of ECP is 50 to 100 times less than that of EDN possibly because of the lack of a positively charged residue at human pancreatic ribonuclease position 122. The calculated isoelectric point (10.8), electronic charge (14.5), and cationic charge distribution of ECP are different from those of EDN but similar to those of MBP, which may account in part for the greater helminthotoxic activity of ECP when compared to EDN. These data suggest that ECP and EDN are derived from a common ancestral ribonuclease gene and that ECP has evolved into a potent helminthotoxin similar in some respects to MBP, while losing much of its ribonuclease activity.     

Dissociation of bovine seminal ribonuclease into catalytically active monomers by selective reduction and alkylation of the intersubunit disulfide bridges.

The hypothesis previously advanced that interchain disulfide bridges link the two identical subunits of bovine seminal ribonuclease BS-1 has been confirmed. The sedimentation rate and the electrophoretic mobility of the protein are not affected by denaturing agents unless thiol reagents are present in the denaturation mixtures. Reduction under controlled conditions results in the immediate cleavage of only 2 disulfide bonds out of 10 percent in the dimeric protein. Under these conditions, and the results do not change when partial reduction is followed by S-alkylation, 30% of the protein dissociates, while the remaining is found to consist of a dimeric species easily dissociable by denaturing agents without addition of thiol reagents. This indicates that the dimeric structure of seminal ribonuclease is maintained not only by disulfide bridges, but also by noncovalent forces. The protein derivative prepared by selective reduction and alkylation has been identified as monomeric bis-S-carboxymethylcysteine-31,32-ribonuclease BS-1. This is on the basis of the characterization of the 14C-labeled S-carboxymethylated peptides isolated from a thermolytic hydrolysate of the derivative prepared with iodo-2-[14C]acetic acid. Monomeric, selectively alkylated ribonuclease BS-1 is stable and catalytically active. The importance of such a derivative is discussed both in the light of the recent studies on the biological actions of seminal ribonuclease and as the fourth component of an experimental system of ribonucleases consisting of two homologous dimers (bovine seminal ribonuclease BS-1 and dimerized bovine pancreatic ribonuclease A) and two homologous monomers (ribonuclease A and the monomeric derivative of ribonuclease BS-1.     

The salt-dependence of a protein-ligand interaction: ion-protein binding energetics

Using the binding of a nucleotide inhibitor (guanosine-3'-monophosphate) to a ribonuclease (ribonuclease Sa) as a model system, we show that the salt-dependence of the interaction arises due to specific ion binding at the site of nucleotide binding. The presence of specific ion-protein binding is concluded from a combination of differential scanning calorimetry and NMR data. Isothermal titration calorimetry data are then fit to determine the energetic profile (enthalpy, entropy, and heat capacity) for both the ion-protein and nucleotide-protein interactions. The results provide insight into the energetics of charge-charge interactions, and have implications for the interpretation of an observed salt-dependence. Further, the presence of specific ion-binding leads to a system behavior as a function of temperature that is drastically different from that predicted from Poisson-Boltzmann calculations.     

Energetics of ribonuclease A catalysis. 3. Temperature dependence of the hydrolysis of cytidine cyclic 2',3'-phosphate

Studies of the temperature dependence of the steady-state kinetics of the ribonuclease A catalyzed hydrolysis of cytidine cyclic 2',3'-phosphate at pH 5.0 are reported. Contributions to the temperature dependence of the apparent Michaelis-Menten parameters from temperature-sensitive protonic equilibria (primarily the coupled protonation/deprotonation of the active-site histidine residues) were included in our analysis. The data were interpreted by employing a transition-state approach. By comparing the temperature dependence of the rate constant for the nonenzymatic hydrolysis of the substrate with the temperature dependence of the enzyme-catalyzed reaction, we obtained values for the enthalpy change, entropy change, and heat capacity change for the interaction of the reaction transition state with the enzyme. These thermodynamic quantities were then interpreted by comparison with corresponding values for the binding of cytidine 2'- and 3'-phosphate to the enzyme. A model is presented for the enzyme-transition-state interaction involving the favorable transfer of a proton from the transition state to a histidine residue at the active site and the formation of hydrogen bonds and van der Waals contacts between the pyrimidine ring of the transition state and the enzyme's binding pocket. These elementary interactions are consistent with the determined values of the enthalpy change and entropy change, as well as earlier reported ionic strength and solvent isotope dependence studies. The Gibbs energy contributions from these elementary interactions have also been estimated, giving a sum approximately equal to the experimentally determined value for the stabilization energy of the enzyme-transition-state complex. The model thus provides an explanation for the magnitude of the approximately 10(10)-fold rate enhancement achieved by this enzyme.     

Chemical modification of ribonuclease A with 4-arsono-2-nitrofluorobenzene

4-Arsono-2-nitrofluorobenzene reacts selectively at the anion binding site of bovine pancreatic ribonuclease A. The major derivative is the inactive 41-(4-arsono-2-nitrophenyl) ribonuclease A (45% yield). Additional products are 1-alpha-(4-arsono-2-nitrophenyl) ribonuclease A (11% yield) which is enzymatically active and the disubstituted, inactive 1,41-bis-(4-arsono-2-nitrophenyl) ribonuclease A (25% yield). 2' (3')-O-Bromoacetyluridine reacts with 41-(4-arsono-2-nitrophenyl) ribonuclease A exclusively at the histidine-12 residue at a rate which is approximately one-fourth the rate observed with the unmodified enzyme. Saturation kinetics are observed and the dissociation constant for the protein-inhibitor complex is 0.096 +/- 0.023 M. The first-order unimolecular decomposition constant for complex breakdown is 8.9 +/- 2.9 X 10(-4) s-1. 2'-Bromoacetamido-2'-deoxyuridine reacts with 41-(4-arsono-2-nitrophenyl) ribonuclease A 25 times more slowly than 2'(3')-O-bromoacetyluridine. Bromoacetate reacts with 41-(4-arsono-2-nitrophenyl) ribonuclease A predominantly at the histidine-119 residue at a rate 45 times less than that found for the unmodified enzyme. The results of the alkylation studies imply that the dianionic arsonate does not occupy the phosphate binding site in the enzyme but is sufficiently proximate to account for a decrease in bromoacetate binding as well as a reduction in the nucleophilic reactivity of histidine-12 and -119. All these effects may be accounted for in terms of a local electrostatic perturbation of the active site region by the arsononitrophenyl group.