PDBsum entry: 2ifv

Hydrolase

PDB id: 2ifv

Contents

Protein chain

175 a.a. *

Metals

_CL ×2

Waters ×240

* Residue conservation analysis

PDB id:

2ifv

Name:

Hydrolase

Title:

Crystal structure of an active site mutant, c473d, of cdc25b phosphatase catalytic domain

Structure:

M-phase inducer phosphatase 2. Chain: a. Fragment: catalytic domain, residues 391-564. Synonym: dual specificity phosphatase cdc25b. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: cdc25b, cdc25hu2. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693.

Resolution:

1.60Å R-factor: 0.179 R-free: 0.192

Authors:

J.Rudolph,G.Buhrman

Key ref:

J.Sohn et al. (2007). Kinetic and structural studies of specific protein-protein interactions in substrate catalysis by Cdc25B phosphatase. Biochemistry, 46, 807-818. PubMed id: 17223702 DOI: 10.1021/bi061257y

Date:

21-Sep-06 Release date: 27-Feb-07

Protein chain

P30305  (MPIP2_HUMAN) -  M-phase inducer phosphatase 2

Seq: 580 a.a.

Struc: 175 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.3.1.3.48 - Protein-tyrosine-phosphatase.
• Reaction: Protein tyrosine phosphate + H₂O = protein tyrosine + phosphate

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

 Gene Ontology (GO) functional annotation 

• Cellular component: intracellular (1 term)
• Biological process: M phase of mitotic cell cycle (2 terms)
• Biochemical function: protein tyrosine phosphatase activity (1 term)

reference

DOI no: 10.1021/bi061257y

Biochemistry 46:807-818 (2007)

PubMed id: 17223702

Kinetic and structural studies of specific protein-protein interactions in substrate catalysis by Cdc25B phosphatase.

J.Sohn, G.Buhrman, J.Rudolph.

ABSTRACT

Using a combination of steady-state and single-turnover kinetics, we probe substrate association, dissociation, and chemistry for the reaction of Cdc25B phosphatase with its Cdk2-pTpY/CycA protein substrate. The rate constant for substrate association for the wild-type enzyme is 1.3 x 10(6) M(-1) s(-1). The rate constant for dissociation is slow compared to the rate constant for phosphate transfer to form the phospho-enzyme intermediate (k2 = 1.1 s(-1)), making Cdk2-pTpY/CycA a sticky substrate. Compared to the wild type, all hotspot mutants of residues at the remote docking site that specifically affect catalysis with the protein substrate (Arg488, Arg492, and Tyr497 on Cdc25B and Asp206 on Cdk2) have greatly slowed rate constants of association (70- to 4500-fold), and some mutants have decreased k2 values compared to that of the wild type. Most dramatically, R492L, despite showing no significant changes in a crystal structure at 2.0 A resolution, has an approximately 100-fold decrease in k2 compared to that of wild-type Cdc25B. The active site C473S mutant binds tightly to and dissociates slowly from Cdk2-pTpY/CycA (Kd = 10 nM, k(off) = 0.01 s(-1)). In contrast, the C473D mutant, despite showing only localized perturbations in the active site at 1.6 A resolution, has a much weaker affinity and dissociates rapidly (Kd of 2 microM, k(off) > 2 s(-1)) from the protein substrate. Overall, we demonstrate that the association of Cdc25B with its Cdk2-pTpY/CycA substrate is governed to a significant extent by the interactions of the remote hotspot residues, whereas dissociation is governed by interactions at the active site.