PDBsum entry: 1c9v

Hydrolase

PDB-id: 1c9v

Asymmetric unit

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Description

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PROCHECK

Protein chain

124 a.a. *

Metal ions

_CL ×2

Waters ×98

* Residue conservation analysis

Tools

Clefts Calculation

Biological unit*, dimer
(*as deduced by PQS)

PDB id:

1c9v

Name:

Hydrolase

Title:

H12a variant of ribonuclease a

Structure:

Ribonuclease a. Chain: a. Engineered: yes. Mutation: yes

Source:

Bos taurus. Cattle. Organism_taxid: 9913. Organ: pancreas. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biological unit:

Dimer (from PQS)

UniProt:

P61823 (RNAS1_BOVIN)

Seq:

150 a.a.

Struc:

124 a.a.*

Key:	PfamA domain
Secondary structure	CATH domain

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme class:

E.C.3.1.27.5   [IntEnz]   [ExPASy]   [KEGG]   [BRENDA]

Reaction:

Endonucleolytic cleavage to nucleoside 3'-phosphates and 3'-phosphooligonucleotides ending in C-P or U-P with 2',3'-cyclic phosphate intermediates.

Resolution:

1.70Å

R-factor:

0.182

Authors:

C.Park,L.W.Schultz,R.T.Raines

Key ref:

C.Park et al. (2001). Contribution of the active site histidine residues of ribonuclease A to nucleic acid binding.. Biochemistry, 40, 4949-4956. [PubMed id: 11305910] [DOI: 10.1021/bi0100182]

Date:

03-Aug-99

Release date:

27-Jun-01

Related entries:

1c9x
h119a ribonuclease a
1c8w
t45g ribonuclease a
3rsd
d121n ribonuclease a
3rsk
k7a, r10a, k66a ribonuclease a
3rsp
p93g ribonuclease a
... plus others (see Header records)

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Clefts

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Key reference

DOI no: 10.1021/bi0100182

Biochemistry 40:4949-4956 (2001)

PubMed id: 11305910

Contribution of the active site histidine residues of ribonuclease A to nucleic acid binding.

C.Park, L.W.Schultz, R.T.Raines.

ABSTRACT

His12 and His119 are critical for catalysis of RNA cleavage by ribonuclease A (RNase A). Substitution of either residue with an alanine decreases the value of k(cat)/K(M) by more than 10(4)-fold. His12 and His119 are proximal to the scissile phosphoryl group of an RNA substrate in enzyme-substrate complexes. Here, the role of these active site histidines in RNA binding was investigated by monitoring the effect of mutagenesis and pH on the stability of enzyme-nucleic acid complexes. X-ray diffraction analysis of the H12A and H119A variants at a resolution of 1.7 and 1.8 A, respectively, shows that the amino acid substitutions do not perturb the overall structure of the variants. Isothermal titration calorimetric studies on the complexation of wild-type RNase A and the variants with 3'-UMP at pH 6.0 show that His12 and His119 contribute 1.4 and 1.1 kcal/mol to complex stability, respectively. Determination of the stability of the complex of wild-type RNase A and 6-carboxyfluorescein approximately d(AUAA) at varying pHs by fluorescence anisotropy shows that the stability increases by 2.4 kcal/mol as the pH decreases from 8.0 to 4.0. At pH 4.0, replacing His12 with an alanine residue decreases the stability of the complex with 6-carboxyfluorescein approximately d(AUAA) by 2.3 kcal/mol. Together, these structural and thermodynamic data provide the first thorough analysis of the contribution of histidine residues to nucleic acid binding.