One site mutation disrupts dimer formation in human DPP-IV proteins
Resource
Journal of Biological Chemistry 279(50): 52338-52345
Journal
Journal of Biological Chemistry
Journal Issue
50
Pages
52338-52345
Date Issued
2004
Date
2004
Author(s)
Abstract
DPP-IV is a prolyl dipeptidase, cleaving the peptide
bond after the penultimate proline residue. It is an important
drug target for the treatment of type II diabetes.
DPP-IV is active as a dimer, and monomeric DPP-IV has
been speculated to be inactive. In this study, we have
identified the C-terminal loop of DPP-IV, highly conserved
among prolyl dipeptidases, as essential for dimer
formation and optimal catalysis. The conserved residue
His750 on the loop contributes significantly for dimer
stability. We have determined the quaternary structures
of the wild type, H750A, and H750E mutant enzymes by
several independent methods including chemical crosslinking,
gel electrophoresis, size exclusion chromatography,
and analytical ultracentrifugation. Wild-type
DPP-IV exists as dimers both in the intact cell and in
vitro after purification from human semen or insect
cells. The H750A mutation results in a mixture of
DPP-IV dimer and monomer. H750A dimer has the same
kinetic constants as those of the wild type, whereas the
H750A monomer has a 60-fold decrease in kcat. Replacement
of His750 with a negatively charged Glu (H750E)
results in nearly exclusive monomers with a 300-fold
decrease in catalytic activity. Interestingly, there is no
dynamic equilibrium between the dimer and the monomer
for all forms of DPP-IVs studied here. This is the
first study of the function of the C-terminal loop as well
as monomeric mutant DPP-IVs with respect to their enzymatic
activities. The study has important implications
for the discovery of drugs targeted to the dimer
interface.
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