Structural changes that accompany the reduced catalytic efficiency of two semisynthetic ribonuclease analogs.

deMel, V.S.J.; Martin, Philip D.; Doscher, Marilynn S.; Edwards, Brian F.P.

doi:10.1016/s0021-9258(18)48486-4

Cited by 31 publications

(2 citation statements)

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“…This deviation is consistent with the semisynthesis having introduced unpredictable structural perturbations. Indeed, x-ray diffractions analyses of the two semisynthetic enzymes revealed many small changes in their structures (de Mel et al, 1992). In contrast, the structure of D121N RNase A is essentially identical to that of the authentic wild-type enzyme (Schultz et al, 1998).…”

Section: Unliganded Enzymesmentioning

confidence: 99%

His ... Asp Catalytic Dyad of Ribonuclease A: Histidine pKa Values in the Wild-Type, D121N, and D121A Enzymes

Quirk

Raines

1999

Biophysical Journal

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Bovine pancreatic ribonuclease A (RNase A) has a conserved His ... Asp catalytic dyad in its active site. Structural analyses had indicated that Asp121 forms a hydrogen bond with His119, which serves as an acid during catalysis of RNA cleavage. The enzyme contains three other histidine residues including His12, which is also in the active site. Here, 1H-NMR spectra of wild-type RNase A and the D121N and D121A variants were analyzed thoroughly as a function of pH. The effect of replacing Asp121 on the microscopic pKa values of the histidine residues is modest: none change by more than 0.2 units. 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. In the presence of the reaction product, uridine 3'-phosphate (3'-UMP), protonation of one active-site histidine residue favors protonation of the other. This finding is consistent with the phosphoryl group of 3'-UMP interacting more strongly with the two active-site histidine residues when both are protonated. Comparison of the titration curves of the unliganded enzyme with that obtained in the presence of different concentrations of 3'-UMP shows that a second molecule of 3'-UMP can bind to the enzyme. Together, the data indicate that the aspartate residue in the His ... Asp catalytic dyad of RNase A has a measurable but modest effect on the ionization of the adjacent histidine residue.

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Section: Unliganded Enzymesmentioning

confidence: 99%

His ... Asp Catalytic Dyad of Ribonuclease A: Histidine pKa Values in the Wild-Type, D121N, and D121A Enzymes

Quirk

Raines

1999

Biophysical Journal

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“…However, the lack of this Asp-121 hydrogenbonded to His-119 does not completely eliminate enzymatic activity. The replacement of Asp-121 by Ala results in a decrease of activity; the kIt/KM is 1.91 mM-1 s-1 for semisynthetic wild-type RNase A and 0.25 mM-1 s-1 for Ala-121 substituted semisynthetic RNase A (deMel et al, 1992). Moreover, the loss of hydrogen bonding capability between Asp-121 and methylated His-119 in semisynthetic RNase A results in neither a change in the pKa of His-119 (Serdijn et al, 1984a, b) nor a total loss of enzymatic activity.…”

Section: Resultsmentioning

confidence: 98%

NMR study of the positions of His-12 and His-119 in the ribonuclease A-uridine vanadate complex

Veenstra

Lee

1994

Biophysical Journal

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The binding of uridine vanadate to ribonuclease A has been investigated by one- and two-dimensional 1H NMR. The homonuclear Nuclear Overhauser and exchange spectroscopy spectrum of the uridine vanadate/RNase A complex exhibits cross peaks between both the C5H and C6H protons of uridine vanadate and the H epsilon 1 proton of His-12 of ribonuclease A. These cross peaks suggest that the H epsilon 1 proton of His-12 is in the vicinity of the uracil base of uridine vanadate, as observed in the crystallographic structure of the uridine vanadate/RNase A complex. However, no cross peaks are observed between the C5H and C6H protons of uridine vanadate and the H epsilon 1 proton of His-119 of ribonuclease A, although they were predicted based upon the distances calculated from coordinates of the crystallographic structure of the complex. These results suggest that there is a significant difference between the positioning of the His-119 side chain in the solution and in the crystallographic structures.

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The crystal structure of recombinant rat pancreatic RNase A

Gupta¹,

Wyns²,

Salunke³

1999

Proteins

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The three-dimensional structure of rat pancreatic RNase A expressed in Escherichia coli was determined. The backbone conformations of certain critical loops are significantly different in this enzyme compared to its bovine counterpart. However, the core structure of rat RNase A is similar to that of the other members of the pancreatic ribonuclease family. The structural variations within a loop bordering the active site can be correlated with the subtle differences in the enzymatic activities of bovine and rat ribonucleases for different substrates. The most significant difference in the backbone conformation was observed in the loop 15-25. This loop incorporates the subtilisin cleavage site which is responsible for RNase A to RNase S conversion in the bovine enzyme. The rat enzyme does not get cleaved under identical conditions. Molecular docking of this region of the rat enzyme in the active site of subtilisin shows steric incompatibility, although the bovine pancreatic ribonuclease A appropriately fits into this active site. It is therefore inferred that the local conformation of the substrate governs the specificity of subtilisin.

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Structural changes that accompany the reduced catalytic efficiency of two semisynthetic ribonuclease analogs.

Cited by 31 publications

References 50 publications

His ... Asp Catalytic Dyad of Ribonuclease A: Histidine pKa Values in the Wild-Type, D121N, and D121A Enzymes

His ... Asp Catalytic Dyad of Ribonuclease A: Histidine pKa Values in the Wild-Type, D121N, and D121A Enzymes

NMR study of the positions of His-12 and His-119 in the ribonuclease A-uridine vanadate complex

The crystal structure of recombinant rat pancreatic RNase A

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