PDBsum entry 1mff

Cytokine

PDB id: 1mff

Contents

Protein chains

114 a.a. *

Waters ×57

* Residue conservation analysis

PDB id:

1mff

Name:

Cytokine

Title:

Macrophage migration inhibitory factor y95f mutant

Structure:

Macrophage migration inhibitory factor. Chain: a, b, c. Synonym: phenylpyruvate tautomerase. Engineered: yes. Mutation: yes

Source:

Mus musculus. House mouse. Organism_taxid: 10090. Gene: mif. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Trimer (from PQS)

Resolution:

2.00Å R-factor: 0.191 R-free: 0.231

Authors:

A.B.Taylor,S.L.Stamps,S.C.Wang,M.L.Hackert,C.P.Whitman

Key ref:

S.L.Stamps et al. (2000). Mechanism of the phenylpyruvate tautomerase activity of macrophage migration inhibitory factor: properties of the P1G, P1A, Y95F, and N97A mutants. Biochemistry, 39, 9671-9678. PubMed id: 10933783 DOI: 10.1021/bi000373c

Date:

19-Oct-98 Release date: 12-Jul-99

Protein chains

P34884  (MIF_MOUSE) -  Macrophage migration inhibitory factor from Mus musculus

Seq: 115 a.a.

Struc: 114 a.a.*

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

Enzyme reactions

• Enzyme class 2: E.C.5.3.2.1 - phenylpyruvate tautomerase.
• Reaction: 3-phenylpyruvate = enol-phenylpyruvate
• Enzyme class 3: E.C.5.3.3.12 - L-dopachrome isomerase.

Pathway:

• Reaction: L-dopachrome = 5,6-dihydroxyindole-2-carboxylate
• Cofactor: Zn(2+)

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

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

reference

DOI no: 10.1021/bi000373c

Biochemistry 39:9671-9678 (2000)

PubMed id: 10933783

Mechanism of the phenylpyruvate tautomerase activity of macrophage migration inhibitory factor: properties of the P1G, P1A, Y95F, and N97A mutants.

S.L.Stamps, A.B.Taylor, S.C.Wang, M.L.Hackert, C.P.Whitman.

ABSTRACT

Phenylpyruvate tautomerase (PPT) has been studied periodically since its activity was first described over forty years ago. In the last two years, the mechanism of PPT has been investigated more extensively because of the discovery that PPT is the same protein as the immunoregulatory cytokine known as macrophage migration inhibitory factor (MIF). The mechanism of PPT is likely to involve general base-general acid catalysis. While several lines of evidence implicate Pro-1 as the general base, the identity of the general acid remains unknown. Crystal structures of MIF with the competitive inhibitor (E)-2-fluoro-p-hydroxycinnamate bound in the active site and that of the protein complexed with the enol form of a substrate, (p-hydroxyphenyl)pyruvate, suggest that Tyr-95 is the only candidate in the vicinity that can function as a general acid catalyst. Although Tyr-95 is nearby the bound inhibitor and substrate, it is not within hydrogen bonding distance of either ligand. In this study, Tyr-95 was mutated to phenylalanine, and the kinetic and structural properties of the Y95F mutant were determined. This alteration produces a fully active enzyme, which shows no significant structural changes in the active site. The results indicate that Tyr-95 does not function as the general acid catalyst in the reaction catalyzed by wild-type PPT. The mechanism of PPT was studied further by constructing and characterizing the kinetic properties of two mutants of Pro-1 (P1G and P1A) and one mutant of Asn-97 (N97A). The mutation of Asn-97, a residue implicated in the binding of the phenolic hydroxy group of the keto and enol isomers of (p-hydroxyphenyl)pyruvate and of (E)-2-fluoro-p-hydroxycinnamate affects only the binding affinity of the inhibitor. However, the mutations of Pro-1 have a profound effect on the values of k(cat) and k(cat)/K(m) and clearly show that Pro-1 is a critical residue in the reaction. The results are discussed in terms of a mechanism in which Pro-1 functions as both the general acid and the general base catalyst.