Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. III. Mutual electrostatic interaction between histidine residues 12 and 119

Markley, John L.; Finkenstadt, William R.

doi:10.1021/bi00687a008

Cited by 45 publications

(34 citation statements)

References 17 publications

(13 reference statements)

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“…The values agree well with results from NMR spectroscopy (88)(89)(90)(91)54). Differences for any given pK a value are within error, especially when recalling that when two pK a 's have similar values, the average value is much better determined from pH-rate profiles than are the individual values (92).…”

Section: Ph-dependence Of Catalysissupporting

confidence: 87%

His···Asp Catalytic Dyad of Ribonuclease A: Structure and Function of the Wild-Type, D121N, and D121A Enzymes

1998

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The sidechains of histidine and aspartate residues form a hydrogen bond in the active sites of many enzymes. In serine proteases, the His⋯Asp hydrogen bond of the catalytic triad is known to contribute greatly to catalysis, perhaps via the formation of a low-barrier hydrogen bond. In bovine pancreatic ribonuclease A (RNase A), the His⋯Asp dyad is composed of His119 and Asp121. Previously, sitedirected mutagenesis was used to show that His119 has a fundamental role-to act as an acid during catalysis of RNA cleavage , J. Am. Chem. Soc. 116,[5467][5468]. Here, Asp121 was replaced with an asparagine or alanine residue. The crystalline structures of the two variants were determined by X-ray diffraction analysis to a resolution of 1.6 Å with an R-factor of 0.18. Replacing Asp121 with an asparagine or alanine residue does not perturb the overall conformation of the enzyme. In the structure of D121N RNase A, N δ rather than O δ of Asn121 faces His119. This alignment in the crystalline state is unlikely to exist in solution because catalysis by the D121N variant is not compromised severely. The steady-state kinetic parameters for catalysis by the wild-type and variant enzymes were determined for the cleavage of uridylyl(3′→5′)adenosine and poly(cytidylic acid), and for the hydrolysis of uridine 2′,3′-cyclic phosphate. Replacing Asp121 decreases the values of k cat /K m and k cat for cleavage by 101-fold (D121N) and 10 2 -fold (D121A). Replacing Asp121 also decreases the values of k cat /K m and k cat for hydrolysis by 10 0.5 -fold (D121N) and 10-fold (D121A), but has no other effect on the pH-rate profiles for hydrolysis. There is no evidence for the formation of a low-barrier hydrogen bond between His119 and either an aspartate or an asparagine residue at position 121. Apparently, the major role of Asp121 is to orient the proper tautomer of His119 for catalysis. Thus, the mere presence of a His⋯Asp dyad in an enzymic active site is not a mandate for its being crucial in effecting catalysis.Keywords catalytic dyad; catalytic triad; low-barrier hydrogen bond; pH-rate profile; ribonuclease A; serine protease; site-directed mutagenesis; X-ray crystallographyThe amino acid motifs available to enzymic catalysts are diverse. Still, many enzymes fall into classes wherein great similarities exist. One well-studied class is that of the serine proteases (1). This class of enzymes has an active-site motif known as the catalytic triad, which is composed of the residues Ser⋯His⋯Asp linked by hydrogen bonds (2,3). † This work was supported by Grant GM44783 (NIH). X-ray data collection and computational facilities were supported by Grant BIR-9317398 (NSF). L.W.S. was supported by postdoctoral fellowship CA69750 (NIH). D.J.Q. was supported by Cellular and Molecular Biology Training Grant GM07215 (NIH).*Author to whom correspondence should be addressed.. ‡ Present address: School of Pharmacy, University of Wisconsin -Madison, Madison, WI 53706. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2...

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Section: Ph-dependence Of Catalysissupporting

confidence: 87%

His···Asp Catalytic Dyad of Ribonuclease A: Structure and Function of the Wild-Type, D121N, and D121A Enzymes

1998

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“…A summary of the titration parameters for the histidyl C O1^H resonances as well as those of other ionizable groups of histatin 5 are given in Table 1. Titration of zinc^histatin solutions prepared at similar pH and concentration resulted in spectra showing a selective shifting of the His-15 resonance compared with the his- [20]. See Section 2 for details.…”

Section: Resultsmentioning

confidence: 99%

“…The pK a and Hill coe¤cients were determined by the method of Markley and Finkenstadt [20] using a Levenberg^Marquardt algorithm for curve-¢tting. Starting values for curve-¢tting parameters were estimated from the resonance positions at the extremes of the pK a range of titrating groups.…”

Section: Estimation Of Pk a Valuesmentioning

confidence: 99%

Zinc and copper bind to unique sites of histatin 5

et al. 2001

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Metal binding has been suggested to be relevant in the antifungal and antibacterial mechanism of histatin 5, a human salivary protein. Proton nuclear magnetic resonance (NMR) spectra were obtained to investigate the specificity of metal binding to the seven histidyl, one aspartyl and one glutamyl amino acid side-chains of histatin 5 in aqueous solutions. Three C O1^H histidyl and the C Q^H glutamyl resonances of histatin 5 were selectively altered in spectra of solutions containing three equivalents of zinc. Copper binding to histatin 5 resulted in a reduced intensity of C L^H aspartyl resonances, while no evidence for calcium binding was found. These results indicate that zinc binding to histatin 5 involves His-15 present within the^H^E^XX^H^z inc binding motif, and copper binding occurs within the N-terminal D^S^H^, ATCUN motif. ß

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“…1 H NMR-pH titration of wild and mutant ribonuclease A yielded microconstants for the catalytic imidazoles of His 12 and His 119 [240]. The microconstants quantitate that (a) the Asp 121 of the catalytic site modulates only slightly the intrinsic basicity of His 119 and (b) the negative cooperativity of His 12 and His 119 in the unliganded enzyme [241] changes to positive cooperativity upon binding the reaction product 3′-UMP [240]. …”

Section: Miscellaneousmentioning

confidence: 99%

Determination of microscopic acid?base parameters from NMR?pH titrations

Szakács

Kraszni

Noszál

2004

Analytical and Bioanalytical Chemistry

106

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The theory and practice of proton microspeciation based on NMR-pH titrations are surveyed. Principles of bi-, tri-, tetra-, and n-protic microequilibrium systems are discussed. Evaluation methods are exemplified by case studies on bi- and tetraprotic biomolecules. Selection criteria and properties of 'reporter' NMR nuclei are described. Literature data on complete microspeciations of small ligands and site-specific basicity characterizations of peptides and proteins are critically reviewed.

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Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. III. Mutual electrostatic interaction between histidine residues 12 and 119

Cited by 45 publications

References 17 publications

His···Asp Catalytic Dyad of Ribonuclease A: Structure and Function of the Wild-Type, D121N, and D121A Enzymes

His···Asp Catalytic Dyad of Ribonuclease A: Structure and Function of the Wild-Type, D121N, and D121A Enzymes

Zinc and copper bind to unique sites of histatin 5

Determination of microscopic acid?base parameters from NMR?pH titrations

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