PDBsum entry 2a0x

Transferase

PDB id: 2a0x

Contents

Protein chain

282 a.a. *

Ligands

SO4 ×2

DIH

Waters ×60

* Residue conservation analysis

PDB id:

2a0x

Name:

Transferase

Title:

Structure of human purine nucleoside phosphorylase h257f mutant

Structure:

Purine nucleoside phosphorylase. Chain: a. Synonym: inosine phosphorylase, pnp. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: np, pnp. Expressed in: escherichia coli. Expression_system_taxid: 562

Biol. unit:

Trimer (from PDB file)

Resolution:

2.28Å R-factor: 0.227 R-free: 0.251

Authors:

A.S.Murkin,W.Shi,V.L.Schramm

Key ref:

A.S.Murkin et al. (2007). Neighboring group participation in the transition state of human purine nucleoside phosphorylase. Biochemistry, 46, 5038-5049. PubMed id: 17407325

Date:

17-Jun-05 Release date: 06-Jun-06

Protein chain

P00491  (PNPH_HUMAN) -  Purine nucleoside phosphorylase from Homo sapiens

Seq: 289 a.a.

Struc: 282 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.2.4.2.1 - purine-nucleoside phosphorylase.
• Reaction:
  1. a purine D-ribonucleoside + phosphate = a purine nucleobase + alpha- D-ribose 1-phosphate
  2. a purine 2'-deoxy-D-ribonucleoside + phosphate = a purine nucleobase + 2-deoxy-alpha-D-ribose 1-phosphate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

Biochemistry 46:5038-5049 (2007)

PubMed id: 17407325

Neighboring group participation in the transition state of human purine nucleoside phosphorylase.

A.S.Murkin, M.R.Birck, A.Rinaldo-Matthis, W.Shi, E.A.Taylor, S.C.Almo, V.L.Schramm.

ABSTRACT

The X-ray crystal structures of human purine nucleoside phosphorylase (PNP) with bound inosine or transition-state analogues show His257 within hydrogen bonding distance of the 5'-hydroxyl. The mutants His257Phe, His257Gly, and His257Asp exhibited greatly decreased affinity for Immucillin-H (ImmH), binding this mimic of an early transition state as much as 370-fold (Km/Ki) less tightly than native PNP. In contrast, these mutants bound DADMe-ImmH, a mimic of a late transition state, nearly as well as the native enzyme. These results indicate that His257 serves an important role in the early stages of transition-state formation. Whereas mutation of His257 resulted in little variation in the PNP x DADMe-ImmH x SO4 structures, His257Phe x ImmH x PO4 showed distortion at the 5'-hydroxyl, indicating the importance of H-bonding in positioning this group during progression to the transition state. Binding isotope effect (BIE) and kinetic isotope effect (KIE) studies of the remote 5'-(3)H for the arsenolysis of inosine with native PNP revealed a BIE of 1.5% and an unexpectedly large intrinsic KIE of 4.6%. This result is interpreted as a moderate electronic distortion toward the transition state in the Michaelis complex with continued development of a similar distortion at the transition state. The mutants His257Phe, His257Gly, and His257Asp altered the 5'-(3)H intrinsic KIE to -3, -14, and 7%, respectively, while the BIEs contributed 2, 2, and -2%, respectively. These surprising results establish that forces in the Michaelis complex, reported by the BIEs, can be reversed or enhanced at the transition state.