The pH Dependence of the Steady State Kinetic Parameters for Staphylococcal Nuclease-catalyzed Hydrolysis of Deoxythymidine-3′-phosphate-5′-p-nitrophenylphosphate in H2O and D2O

Dunn, Ben M.; DiBello, Carlo; Anfinsen, Christian B.

doi:10.1016/s0021-9258(19)43731-9

Cited by 40 publications

(19 citation statements)

References 21 publications

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“…All assays in the present study were carried out at 300 mM ionic strength, contributed mainly by KC1 and buffer. As a result, the pAa values obtained here are consistently higher than those observed by Dunn et al (1973) at about 50-100 trations, kcu is the dominant term, whereas at very low substrate concentrations, kM/Km describes the enzyme rate vs substrate concentration function. Km is the ratio of kM and kM/Km and does not have to be defined to know km/Km precisely.…”

Section: Resultssupporting

confidence: 44%

“…In the present paper, we have made use of a nuclease variant produced by site-directed mutagensis to investigate the effect of changing Tyr-85 to Phe (nuclease Y85F). We have found that the mutant lacks the pATa of 9.15 observed by Dunn et al (1973). This result suggests that the p at 9.15 belongs 3 'H NMR studies of nuclease wt (Dr. Eldon L. Ulrich, personal communication) indicate that chemical shift coalescence of amino acids of the same type (as would happen in a denatured protein without significant secondary or tertiary structure) does not occur below pH 11.5 in D20 and 0.3 M KC1.…”

mentioning

confidence: 57%

“…to be hydrolysis of the phosphodiester bond), the titration of this residue should be observable in the k^/Km vs pH profile. Dunn et al (1973) observed a bell-shaped dependence of kcat/Km with pvalues at 8.43 and 9.15 on following hydrolysis of the DNA analog thymidine 3'-phosphate 5'-{pnitrophenyl phosphate) to the product thymidine 3'-phosphate (Figure 2). They observed only one pA^a at 9.01 in the kv s pH profile.…”

mentioning

confidence: 96%

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Staphylococcal nuclease active-site amino acids: pH dependence of tyrosines and arginines by carbon-13 NMR and correlation with kinetic studies

Grissom

Markley²

1989

Biochemistry

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The pH and temperature dependence of the kinetic parameters of staphylococcal nuclease (EC 3.1.4.7) have been examined with three p-nitrophenyl phosphate containing DNA analogues that vary as to 3'-substituent. With wild-type (Foggi variant) (nuclease wt) and the substrates thymidine 3'-phosphate 5'-(p-nitrophenyl phosphate) (PNPdTp), thymidine 3'-methylphosphonate 5'-(p-nitrophenyl phosphate) (PNPdTp*Me), and thymidine 5'-(p-nitrophenyl phosphate) (PNPdT), kcat remains nearly constant at 13 min-1. However, kcat/Km with nuclease wt varies considerably: 413, 13, and 0.52 mM-1 min-1 with PNPdTp, PNPdTp*Me, and PNPdT, respectively. When tyrosine-85 is changed to phenylalanine (nuclease Y85F) by site-directed mutagenesis, kcat is unchanged at about 13 min-1, except with PNPdTp where it drops to 1 min-1. With nuclease Y85F, kcat/Km is 19.5 and 25 mM-1 min-1 with PNPdTp and PNPdTp*Me, respectively. With PNPdTp as the substrate, a bell-shaped kcat/Km vs pH profile is seen with pKa values at 8.94 and 9.67 in 0.3 M KCl and H2O. The pKa at 9.67 disappears, and a new pKa appears at 10.1 when tyrosine-85 is changed to phenylalanine (nuclease Y85F) or when the substrate 3'-phosphomonoester is changed to a 3'-methylphosphonate (PNPdTp*Me). This suggests that the inflection in kcat/Km with pKa at 9.67 arises from ionization of tyrosine-85, which hydrogen bonds to the divalent 3'-phosphomonoester of substrates with this substituent.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultssupporting

confidence: 44%

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confidence: 57%

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confidence: 96%

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Staphylococcal nuclease active-site amino acids: pH dependence of tyrosines and arginines by carbon-13 NMR and correlation with kinetic studies

Grissom

Markley²

1989

Biochemistry

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“…The enzymatic activity of the D21E mutant of staphylococcal nuclease was measured before and after all NMR experiments by observing the absorbance increase at 260 nm as DNA is hydrolyzed (Cuatrecasas et al, 1967a,b). Protein concentration was determined by absorbance at 280 nm (e01% = 0.93) at neutral pH (Dunn et al, 1973;Tucker et al, 1978Tucker et al, , 1979 or in the presence of nucleotides by the method of Bradford (1976) with the D21E mutant staphylococcal nuclease as the standard. The specific activity of the enzyme was found to be 1.5 units/mg, and the enzyme retained at least 94% of its activity after prolonged NMR studies.…”

Section: Methodsmentioning

confidence: 99%

NMR Docking of a Substrate into the X-ray Structure of the Asp-21 .fwdarw. Glu Mutant of Staphylococcal Nuclease

Weber

Libson²,

Gittis³

et al. 1994

Biochemistry

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To understand the structural basis of the 1500-fold decrease in catalytic activity of the D21E mutant of staphylococcal nuclease in which an aspartate ligand of the essential Ca2+ has been enlarged to glutamate, the conformation of the enzyme-bound substrate dTdA has been determined by NMR methods and has been docked into the X-ray structure of the D21E mutant (Libson, A. M., Gittis, A.G., & Lattman, E. E. Biochemistry, preceding paper in this issue) based on distances from the bound metal ion to dTdA and on intermolecular nuclear Overhauser effects from assigned aromatic proton resonances of Tyr-85, Tyr-113, and Tyr-115 to proton resonances of dTdA, using energy minimization to relieve small overlaps. Like the wild-type enzyme, the D21E mutant forms binary E-M and E-S and ternary E-M-S complexes with Ca2+, Mn2+, Co2+, and La3+. D21E enhances the paramagnetic effects of Co2+ on 1/T1 and 1/T2 of the phosphorus and on 1/T1 of four proton resonances of dTdA, and these effects are abolished by the binding of the competitive inhibitor 3',5'-pdTp. From the paramagnetic effects of enzyme-bound Co2+ on 1/T1 of phosphorus and protons, with the use of a correlation time of 1.1 ps based on 1/T1 values at 250 and 600 MHz, five metal-nucleus distances and 11 lower limit metal-nucleus distances have been calculated. The Co2+ to 31P distance of 4.1 +/- 0.9 A agrees with that found on the wild-type enzyme (Weber, D. J., Mullen, G. P., & Mildvan, A. S. (1991) Biochemistry 30, 7425-7437) and indicates at least 18% inner sphere phosphate coordination. Fourteen interproton distances and 109 lower limit interproton distances in dTdA in the ternary D21E-La(3+)-dTdA complex were determined by NOESY spectra at 50-, 100-, and 200-ms mixing times. Both the metal-nucleus and interproton distances were necessary to compute a narrow range of conformations for enzyme-bound dTdA. As on the wild-type enzyme, the conformation of dTdA on the D21E mutant is highly extended, with high-anti C-2' endo conformations for the individual nucleosides. However, significant conformational differences are found in the torsional angles chi of dA (delta chi = 49 +/- 3 degrees), in gamma of dT (delta gamma = 108 +/- 30 degrees) and in zeta of dT (delta zeta = 124 +/- 38 degrees).(ABSTRACT TRUNCATED AT 400 WORDS)

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“…Water has been proposed as the attacking nucleophile in the mechanism of the nucleotidyl transfer enzyme, staphylococcal nuclease. The calcium ion may increase the nucleophilicity of a bound water molecule, and the metal-bound water is also in a position to displace the poorer of the two possible leaving groups Dunn, di Bello & Anfinsen 1973;Mildvan 1974). In acotinase, a water ligand substitution on the enzyme-bound iron provides the driving force for a conformational change required by the enzyme (Glusker 1968).…”

Section: (I) Internal Watermentioning

confidence: 99%

The organization and function of water in protein crystals

Finney

1977

Phil. Trans. R. Soc. Lond. B

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Dry proteins are dead, or at best asleep. Substitution of D2O can drastically alter biological activity. Water is thus essential in maintaining the structural integrity of biologically active macromolecules, and is implicated in their functioning. Such water may occupy a range of dynamical states, from being strongly bound and localized, to more labile and 'liquid-like'. Spatially ordering the macromolecules aids the search for the more localized water molecules. For example, diffraction experiments on singly crystals can resolve 'bound' water molecules within a protein molecule--ofter at active sites, coordinated to metals or ions. Less precise information is obtained on the partially occupied external water sites, which are of importance to the folding and dynamics of the biomolecule. Orientation of fibrous molecules increases the information obtainable from n.m.r. experiments. Combination of other experimental results on disordered aggregates (e.g. in solution) with chemical and structural data on the macromolecule and water itself yields useful, if circumstantial, information. Statistical and computer techniques may help to elucidate the complex nature of water-protein interactions, and to interpret the results of more complex experiments.

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The pH Dependence of the Steady State Kinetic Parameters for Staphylococcal Nuclease-catalyzed Hydrolysis of Deoxythymidine-3′-phosphate-5′-p-nitrophenylphosphate in H2O and D2O

Cited by 40 publications

References 21 publications

Staphylococcal nuclease active-site amino acids: pH dependence of tyrosines and arginines by carbon-13 NMR and correlation with kinetic studies

Staphylococcal nuclease active-site amino acids: pH dependence of tyrosines and arginines by carbon-13 NMR and correlation with kinetic studies

NMR Docking of a Substrate into the X-ray Structure of the Asp-21 .fwdarw. Glu Mutant of Staphylococcal Nuclease

The organization and function of water in protein crystals

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