PDBsum entry 1ay0

Transferase

PDB id: 1ay0

Contents

Protein chains

678 a.a. *

Ligands

TPP ×2

Metals

_CA ×2

* Residue conservation analysis

PDB id:

1ay0

Name:

Transferase

Title:

Identification of catalytically important residues in yeast transketolase

Structure:

Transketolase. Chain: a, b. Engineered: yes. Mutation: yes

Source:

Saccharomyces cerevisiae. Baker's yeast. Organism_taxid: 4932. Strain: h402. Gene: tkl1. Expressed in: saccharomyces cerevisiae. Expression_system_taxid: 4932

Biol. unit:

Homo-Dimer (from PDB file)

Resolution:

2.60Å R-factor: 0.193 R-free: 0.263

Authors:

C.Wikner,U.Nilsson,L.Meshalkina,C.Udekwu,Y.Lindqvist,G.Schneider

Key ref:

C.Wikner et al. (1997). Identification of catalytically important residues in yeast transketolase. Biochemistry, 36, 15643-15649. PubMed id: 9398292 DOI: 10.1021/bi971606b

Date:

13-Nov-97 Release date: 13-May-98

Protein chains

P23254  (TKT1_YEAST) -  Transketolase 1 from Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Seq: 680 a.a.

Struc: 678 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.2.2.1.1 - transketolase.
• Reaction: D-sedoheptulose 7-phosphate + D-glyceraldehyde 3-phosphate = aldehydo-D- ribose 5-phosphate + D-xylulose 5-phosphate
• Cofactor: Thiamine diphosphate

Thiamine diphosphate (Bound ligand (Het Group name = TPP) corresponds exactly)

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

reference

DOI no: 10.1021/bi971606b

Biochemistry 36:15643-15649 (1997)

PubMed id: 9398292

Identification of catalytically important residues in yeast transketolase.

C.Wikner, U.Nilsson, L.Meshalkina, C.Udekwu, Y.Lindqvist, G.Schneider.

ABSTRACT

The possible roles of four histidine residues in the active site of yeast transketolase were examined by site-directed mutagenesis. Replacement of the invariant His69 with alanine yielded a mutant enzyme with 1.5% of the specific activity of the wild-type enzyme and with an increased KM for the donor. This residue is located at the bottom of the substrate cleft close to the C1 hydroxyl group of the donor substrate, and the side chain of His69 might be required for recognition of this hydroxyl group and possibly for maintenance of the proper orientation of the reaction intermediate, (alpha, beta-dihydroxyethyl)thiamin diphosphate. Amino acid replacements of His481 by alanine, serine, and glutamine resulted in mutant enzymes with significantly increased KM values for the donor substrate and specific activities of 4.4%, 1.9%, and 5.5% of the wild-type enzyme. The kinetic data suggest that this residue, although close to the C2 carbonyl oxygen of the substrate, is not absolutely required for stabilization of the negative charge that develops at this oxygen in the transition state. This points toward the 4'-NH2 group of the pyrimidine ring of thiamin diphosphate as the major source of charge stabilization. Mutations at positions His30 and His263 result in mutant enzymes severely impaired in catalytic activity (1.5% and less of the activity of wild-type transketolase). The KM value for the donor substrate was increased for the His30Ala mutant but remained unchanged in the His263Ala enzyme. The side chains of both residues interact with the C3 hydroxyl group of the donor substrate, and the results indicate that the two residues act in concert during proton abstraction of the C3 hydroxyl proton during catalysis.