The contribution of noncatalytic phosphate-binding subsites to the mechanism of bovine pancreatic ribonuclease A

The enzymatic catalysis of polymeric substrates such as proteins, polysaccharides or nucleic acids requires precise alignment between the enzyme and the substrate regions flanking the region occupying the active site. In the case of ribonucleases, enzyme-substrate binding may be directed by electrostatic interactions between the phosphate groups of the RNA molecule and basic amino acid residues on the enzyme. Specific interactions between the nitrogenated bases and particular amino acids in the active site or adjacent positions may also take place. The substrate-binding subsites of ribonuclease A have been characterized by structural and kinetic studies. In addition to the active site (p1), the role of other noncatalytic phosphate-binding subsites in the correct alignment of the polymeric substrate has been proposed. p2 and p0 have been described as phosphate-binding subsites that bind the phosphate group adjacent to the 3' side and 5' side, respectively, of the phosphate in the active site. In both cases, basic amino acids (Lys-7 and Arg-10 in p2, and Lys-66 in p0) are involved in binding. However, these binding sites play different roles in the catalytic process of ribonuclease A. The electrostatic interactions in p2 are important both in catalysis and in the endonuclease activity of the enzyme, whilst the p0 electrostatic interaction contributes only to binding of the RNA.     

A new remote subsite in ribonuclease A

The interaction between bovine pancreatic ribonuclease A (RNase A) and its RNA substrate extends beyond the scissile bond. Enzymic subsites interact with the bases and the phosphoryl groups of a bound substrate. We evaluated the four cationic residues closest to known subsites for their abilities to interact with a bound nucleic acid. Lys-37, Arg-39, Arg-85, and Lys-104 were replaced individually by an alanine residue, and the resulting enzymes were assayed as catalysts of poly(cytidylic acid) (poly(C)) cleavage. The values of Km and kcat/Km for poly(C) cleavage were affected only by replacing Arg-85. Moreover, the contribution of Arg-85 to the binding of the ground state and the transition state was uniform---Km increased by 15-fold and kcat/Km decreased by 10-fold. The contribution of Arg-85 to binding was also apparent in the values of Kd for complexes with oligonucleotides of different length. This contribution was dependent on salt concentration, as expected from a coulombic interaction between a cationic side chain and an anionic phosphoryl group. Together, these data indicate that Arg-85 interacts with a particular phosphoryl group of a bound nucleic acid. We propose that Arg-85 comprises a new distal subsite in RNase A---the P(-1) subsite.     

The role of non-catalytic binding subsites in the endonuclease activity of bovine pancreatic ribonuclease A

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 (Parés, X., Nogués, 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 (p2) adjacent to the catalytic center has been analyzed in p2 chemically modified ribonuclease A or by means of site-directed mutagenesis. In both cases deletion of p2 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.     

Protein components contribute to active site architecture for eukaryotic ribonuclease P

In eukaryotes, ribonuclease P (RNase P) requires both RNA and protein components for catalytic activity. The eukaryotic RNase P RNA, unlike its bacterial counterparts, does not possess intrinsic catalytic activity in the absence of holoenzyme protein components. We have used a sensitive photoreactive cross-linking assay to explore the substrate-binding environment for different eukaryotic RNase P holoenzymes. Protein components from the Tetrahymena thermophila and human RNase P holoenzymes form specific products in photoreactions containing [4-thio]-uridine-labeled pre-tRNAGln. The HeLa RNase P RNA in neither the presence nor the absence of holoenzyme protein components formed cross-link products to the pre-tRNAGln probe. Parallel photo-cross-linking experiments with the Escherichia coli RNase P holoenzyme revealed that only the bacterial RNase P RNA forms specific substrate photoadducts. A protein-rich active site for the eukaryotic RNase P represents one unique feature that distinguishes holoenzyme organization between bacteria and eukaryotes.     

The compactness of ribonuclease A and reduced ribonuclease A

This study reports on a patient with Leigh syndrome with a T-to-C mutation at nucleotide 8993 of mitochondrial deoxyribonucleic acid (T8993C). The authors reviewed 10 Leigh syndrome patients, including ours, with T8993C. Compared with 18 reported patients with Leigh syndrome caused by a T-to-G mutation at nucleotide 8993 (T8993G), Leigh syndrome with T8993C was characterized by a significantly higher frequency of ataxia (P < 0.01). None of the reviewed T8993C-associated Leigh syndrome patients had retinitis pigmentosa, which is one of the characteristic findings in Leigh syndrome with T8993G. The milder symptoms of T8993C-Leigh syndrome can be explained by the milder complex V dysfunction; however, the higher frequency of ataxia in T8993C-Leigh syndrome requires more study.     

Coulombic forces in protein-RNA interactions: binding and cleavage by ribonuclease A and variants at Lys7, Arg10, and Lys66

The interactions between bovine pancreatic ribonuclease A (RNase A) and its RNA substrate extend beyond the scissile P-O5' bond. Enzymic subsites interact with the bases and phosphoryl groups of the bound substrate. Those residues interacting with the phosphoryl group comprise the P0, P1, and P2 subsites, with the scissile bond residing in the P1 subsite. Here, the function of the P0 and P2 subsites of RNase A is characterized in detail. Lys66 (P0 subsite) and Lys7 and Arg10 (P2 subsite) were replaced with alanine residues. Wild-type RNase A and the K66A, K7A/R10A, and K7A/R10A/K66A variants were evaluated as catalysts for the cleavage of poly(cytidylic acid) [poly(C)] and for their abilities to bind to single-stranded DNA, a substrate analogue. The values of kcat and Km for poly(C) cleavage were affected by altering the P0 and P2 subsites. The kcat/Km values for poly(C) cleavage by the K66A, K7A/R10A, and K7A/R10A/K66A variants were 3-fold, 60-fold, and 300-fold lower, respectively, than that of wild-type RNase A. These values indicate that the P0 and P2 subsites contribute 0.70 and 2.46 kcal/mol, respectively, to transition-state binding. Binding experiments indicate that the P0 and P2 subsites contribute 0.92 and 1.21 kcal/mol, respectively, to ground-state binding. Thus, the P0 subsite makes a uniform contribution toward binding the ground state and the transition state, whereas the P2 subsite differentiates, binding more tightly to the transition state than to the ground state. In addition, nucleic acid binding to wild-type RNase A is strongly dependent on NaCl concentration, but this dependence is diminished upon alteration of the P0 or P2 subsite. The logarithm of Kd is a linear function of the logarithm of [Na+] over the range 0.018 M 相似文献   

Binding of different divalent cations to the active site of avian sarcoma virus integrase and their effects on enzymatic activity

Retroviral integrases (INs) contain two known metal binding domains. The N-terminal domain includes a zinc finger motif and has been shown to bind Zn2+, whereas the central catalytic core domain includes a triad of acidic amino acids that bind Mn2+ or Mg2+, the metal cofactors required for enzymatic activity. The integration reaction occurs in two distinct steps; the first is a specific endonucleolytic cleavage step called "processing," and the second is a polynucleotide transfer or "joining" step. Our previous results showed that the metal preference for in vitro activity of avian sarcoma virus IN is Mn2+ > Mg2+ and that a single cation of either metal is coordinated by two of the three critical active site residues (Asp-64 and Asp-121) in crystals of the isolated catalytic domain. Here, we report that Ca2+, Zn2+, and Cd2+ can also bind in the active site of the catalytic domain. Furthermore, two zinc and cadmium cations are bound at the active site, with all three residues of the active site triad (Asp-64, Asp-121, and Glu-157) contributing to their coordination. These results are consistent with a two-metal mechanism for catalysis by retroviral integrases. We also show that Zn2+ can serve as a cofactor for the endonucleolytic reactions catalyzed by either the full-length protein, a derivative lacking the N-terminal domain, or the isolated catalytic domain of avian sarcoma virus IN. However, polynucleotidyl transferase activities are severely impaired or undetectable in the presence of Zn2+. Thus, although the processing and joining steps of integrase employ a similar mechanism and the same active site triad, they can be clearly distinguished by their metal preferences.     

Introduction: the ribonuclease A superfamily

In this multi-author issue several aspects of the ribonuclease A superfamily are reviewed. This superfamily can be subdivided in a number of mammalian and other vertebrate ribonuclease families. In the introduction chapter the titles of the other contributions are presented. There is little uniformity in the nomenclature of ribonucleases, caused in part by gene duplications, which have occurred independently in several mammalian lineages, and which are nice examples for explaining orthology and paralogy in molecular evolution.     

Mapping of the active site of recombinant human erythropoietin

Recombinant human erythropoietin (rHuEPO) variants have been constructed to identify amino acid residues important for biological activity. Immunoassays were used to determine the effect of each mutation on rHuEPO folding. With this strategy, we could distinguish between mutations that affected bioactivity directly and those that affected bioactivity because the mutation altered rHuEPO conformation. Four regions were found to be important for bioactivity: amino acids 11 to 15, 44 to 51, 100 to 108, and 147 to 151. EPO variants could be divided into two groups according to the differential effects on EPO receptor binding activity and in vitro biologic activity. This suggests that rHuEPO has two separate receptor binding sites. Mutations in basic residues reduced the biologic activity, whereas mutations in acidic residues did not. This suggests that electrostatic interactions between rHuEPO and the human EPO receptor may involve positive charges on rHuEPO.     

A study of the alkaline mesentericopeptidase active site by means of peptide chloromethyl ketones

The kinetics of inactivation of alkaline mesentericopeptidase was studied using chloromethyl ketone derivatives of amino acids and peptides. It was shown that Tos-LysCH2Cl and Tos-PheCH2Cl did not influence the enzyme activity, while the inhibitory effect of Cbz-Ala-Gly-PheCH2Cl was 35 times that of Cbz-Ala-PheCH2Cl. The dependence of the pseudo-first order rate constant of the enzyme inactivation by Cbz-Ala-Gly-PheCH2Cl on pH and temperature indicated that a group with a pK of 6.59 and deltaHi of 7.7 kcal/mol was the site of the inhibitor's attack. Amino acid analysis of the modified totally inactive enzyme revealed a definite loss of histidine and after performic acid oxidation a recovery of 3-carboxymethyl histidine. The whole set of experimental data is convincing evidence on behalf of a selective alkylation of the N-3 of the active site histidine after treatment with Cbz-Ala-PheCH2Cl and Cbz-Ala-Gly-PheCH2Cl. Alkaline mesenteriocopeptidase possesses an extended active site and only a peptide chloromethyl ketone, covering a determined sequence of theenzyme molecule (S3, S2, S1, S'1, S'2, S'3 ...) is able to provide effective inhibition. The values of the inactivation constant (kinact) for Cbz-Ala-PheCH2Cl and Cbz-Ala-Gly-PheCH2Cl are close to the corresponding values reported for subtilisin Amylosacchariticus.     

The active site specificity of the Yersinia protein-tyrosine phosphatase

Previous studies have shown that B700, an albumin-like murine melanoma antigen, has a human homologue termed H700. Polyclonal antibodies to B700 also bind to all cultured human, swine and hamster melanoma cells, suggesting that B700 is a "pan-melanoma" antigen. The objects of this investigation were: (a) to determine if 2-3-3, a monoclonal antibody to B700, can be used to identify human melanomas in formalin-fixed, paraffin-embedded tissues, and (b) to determine the specificity and potential diagnostic value of 2-3-3. Forty-eight of the 49 human melanomas, including spindle melanoma cells, stained positively, as did five of the eight pigmented naevi including cellular spindle naevi. Twenty-six of the 32 human non-melanomatous lesions were negative for 2-3-3 staining (weakly positive on one breast carcinoma and positive on five neural tumours). These results indicate that 2-3-3, a monoclonal antibody to the mouse melanoma antigen B700, can be used to identify H700 in archival specimens. 2-3-3 may have an advantage over HMB45, which is the most commonly used antibody for melanoma diagnosis, because of its immunoreactivity with spindle melanocytic lesions. Antibodies to B700 may prove to be a useful adjunct in the diagnosis of human melanoma and related lesions.     

Homologies in the active site regions of lactate dehydrogenases

The persistence of viral DNA in BHK-21 cells abortively infected with human adenovirus type 12 has been investigated using reassociation kinetics. No indication of an increase in the amount of viral DNA per cell has been found. On the contrary, the amount of intracellular viral DNA sequences decreases rapidly after infection. Thus, free adenovirus type 12 DNA does not replicate in BHK-21 cells. The influence of the multiplicity of infection on the amount of persisting adenovirus type 12 DNA has also been explored. The viral DNA sequences persisting in four lines of hamster cells transformed in vitro by adenovirus type 12 at various multiplicities of infection have been quantitated and mapped by reassociation kinetics experiments using restriction endonuclease fragments of 3H-labeled adenovirus type 12 DNA. All the EcoRI restriction nuclease fragments of the adenovirus type 12 genome are represented in each of the four cell lines. Individual fragments of the viral genome are represented in multiple copies in non-equimolar amounts.     

A theoretical analysis of the binding of methyl derivatives of uracil at the contact portion of the active center of ribonuclease S

Increased entry of deoxy[3H]cytidine begins at about 12h after addition of phytohaemagglutinin to peripheral pig lymphocyte cultures, and is accompanied by a parallel stimulation of deoxycytidine kinase up to the beginning of DNA synthesis at 24h. The increased deoxycytidine uptake is characterized by an increase in Vmax. without alteration of the apparent Km (0.7 +/- 0.11 muM). Although the entries of both nucleosides are promoted at the same time, the stimulation of deoxycytidine uptake is less than that of thymidine, and the two nucleosides are transported by separate systems. In addition to deoxycytidien kinase, the synthesis of deoxycytidylate deaminase and thymidylate synthetase are stimulated after addition of phytohaemagglutinin, but to a lesser extent than that of thymidine kinase. The importance of the latter enzyme in forming dTMP, and of thymidylate kinase in providing dTTP, is discussed.     

Conserved features in the active site of nonhomologous serine proteases

BACKGROUND: Serine protease activity is critical for many biological processes and has arisen independently in a few different protein families. It is not clear, though, the degree to which these protease families share common biochemical and biophysical properties. We have used a computer program to study the properties that are shared by four serine protease active sites with no overall structural or sequence homology. The program systematically compares the region around the catalytic histidines from the four proteins with a set of noncatalytic histidines, used as controls. It reports the three-dimensional locations and level of statistical significance for those properties that distinguish the catalytic histidines from the noncatalytic ones. The method of analysis is general and can be applied easily to other active sites of interest. RESULTS: As expected, some of the reported properties correspond to previously known features of the serine protease active site, including the catalytic triad and the oxyanion hole. Novel properties are also found, including the spatial distribution of charged, polar, and hydrophobic groups arranged to stabilize the catalytic residues, and a relative abundance of some residues (Val, Tyr, Leu, and Gly) around the active site. CONCLUSIONS: Our findings show that in addition to some properties common to all the proteases examined, there are a set of preferred, but not required, properties that can be reliably observed only by aligning the sites and comparing them with carefully selected statistical controls.     

Automated docking of glucosyl disaccharides in the glucoamylase active site

To better understand the molecular basis of glucomylase selectivity, low-energy conformers of glucosyl disaccharides obtained from relaxed-residue conformational mapping were flexibly docked into the glucoamylase active site using AutoDock 2.2. This procedure ensures that significant conformational space is searched and can produce bound structures comparable to those obtained by protein crystallography. alpha-Linked glucosyl disaccharides except alpha,alpha-trehalose dock easily into the active site while exclusively beta-linked disaccharides do not, explaining why only the former are glucoamylase substrates. The optimized docking modes are similar at the nonreducing end of the different substrates. Individual atomic energies of intermolecular interaction allow the definite identification of key hydroxyl groups for each substrate. This approach confirmed the versatility of the second subsite of the glucoamylase active site in binding different substrates.     

Effect of the chaperone-like alpha-crystallin on the refolding of lysozyme and ribonuclease A

This article is a critical review of the evolution of diagnostic criteria for anorexia nervosa, bulimia nervosa, and binge eating disorder. The shortcomings of the current diagnostic criteria for all three disorders are addressed, as are the primary nosological challenges facing the field.     

Kinetic mechanism of active site non-equivalence in transketolase

The two-step mechanism of coenzyme (TDP) binding to apotransketolase has been examined by kinetic modeling, and the rate and equilibrium constants for each binding step for two active sites have been determined. The dissociation constants for the primary fast binding step and the forward rate constants for the secondary slow binding step have been shown to be similar for two active sites. The backward rate constants for the secondary binding step are different for two active sites, providing the kinetic mechanism of their non-equivalence in TDP binding.     

The structure of the MnIIADP-nitrate-lyxose complex at the active site of hexokinase

The coordination scheme of Mn2+ in the hexokinase-MnIIADP-nitrate-lyxose complex has been determined by electron paramagnetic resonance (EPR) spectroscopy with 17O-enriched ligands. Nitrate binds to the active site of hexokinase when MnIIADP and a sugar substrate or analogue are present. The binding of nitrate enhances inhibition by glucose when ADP is present and narrows the EPR signals of the enzyme-bound MnIIADP complex in the presence of sugar substrates or analogues. Experiments using regiospecifically 17O-enriched ADP, 17O-enriched nitrate, and 17O-enriched water establish the coordination scheme of Mn2+. The EPR experiments show that ADP is a beta-monodentate ligand and that nitrate binds directly to Mn2+. Four water molecules complete the coordination sphere of the enzyme-bound Mn2+. The dissociation constant (Kd approximately 8 mM) of nitrate for the complex with enzyme, MnIIADP, and lyxose was obtained from titration experiments. These results suggest that nitrate-stabilized, dead-end complexes of hexokinase may be useful in stabilizing the closed conformation of this "hinge-bending" enzyme for crystallographic experiments.