PDBsum entry 1eid

Hydrolase

PDB id: 1eid

Contents

Protein chain

124 a.a. *

Waters ×142

* Residue conservation analysis

PDB id:

1eid

Name:

Hydrolase

Title:

Crystal structure of f120g mutant of bovine pancreatic 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.

Biol. unit:

Dimer (from PQS)

Resolution:

1.40Å R-factor: 0.210 R-free: 0.265

Authors:

E.Chatani,R.Hayashi,H.Moriyama,T.Ueki

Key ref:

E.Chatani et al. (2002). Conformational strictness required for maximum activity and stability of bovine pancreatic ribonuclease A as revealed by crystallographic study of three Phe120 mutants at 1.4 A resolution. Protein Sci, 11, 72-81. PubMed id: 11742124

Date:

25-Feb-00 Release date: 13-Feb-02

Protein chain

P61823  (RNAS1_BOVIN) -  Ribonuclease pancreatic from Bos taurus

Seq: 150 a.a.

Struc: 124 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.4.6.1.18 - pancreatic ribonuclease.
• Reaction:
  1. an [RNA] containing cytidine + H2O = an [RNA]-3'-cytidine- 3'-phosphate + a 5'-hydroxy-ribonucleotide-3'-[RNA]
  2. an [RNA] containing uridine + H2O = an [RNA]-3'-uridine-3'-phosphate + a 5'-hydroxy-ribonucleotide-3'-[RNA]

Protein Sci 11:72-81 (2002)

PubMed id: 11742124

Conformational strictness required for maximum activity and stability of bovine pancreatic ribonuclease A as revealed by crystallographic study of three Phe120 mutants at 1.4 A resolution.

E.Chatani, R.Hayashi, H.Moriyama, T.Ueki.

ABSTRACT

The replacement of Phe120 with other hydrophobic residues causes a decrease in the activity and thermal stability in ribonuclease A (RNase A). To explain this, the crystal structures of wild-type RNase A and three mutants--F120A, F120G, and F120W--were analyzed up to a 1.4 A resolution. Although the overall backbone structures of all mutant samples were nearly the same as that of wild-type RNase A, except for the C-terminal region of F120G with a high B-factor, two local conformational changes were observed at His119 in the mutants. First, His119 of the wild-type and F120W RNase A adopted an A position, whereas those of F120A and F120G adopted a B position, but the static crystallographic position did not reflect either the efficiency of transphosphorylation or the hydrolysis reaction. Second, His119 imidazole rings of all mutant enzymes were deviated from that of wild-type RNase A, and those of F120W and F120G appeared to be "inside out" compared with that of wild-type RNase A. Only approximately 1 A change in the distance between N(epsilon2) of His12 and N(delta1) of His119 causes a drastic decrease in k(cat), indicating that the active site requires the strict positioning of the catalytic residues. A good correlation between the change in total accessible surface area of the pockets on the surface of the mutant enzymes and enthalpy change in their thermal denaturation also indicates that the effects caused by the replacements are not localized but extend to remote regions of the protein molecule.