A conserved hydrogen-bond network in the catalytic centre of animal glutaminyl cyclases is critical for catalysis
Resource
Biochemistry Journal, 411, 181-190
Journal
Biochemical Journal
Pages
181-190
Date Issued
2008
Date
2008
Author(s)
Huang, Kai-Fa
Wang, Yu-Ruei
Chang, En-Cheng
Chou, Tsung-Lin
Wang, Andrew H.-J.
Abstract
QCs (glutaminyl cyclases; glutaminyl-peptide cyclotransferases, EC 2.3.2.5) catalyse N-terminal pyroglutamate formation in numerous bioactive peptides and proteins. The enzymes were reported to be involved in several pathological conditions such as amyloidotic disease, osteoporosis, rheumatoid arthritis and melanoma. The crystal structure of human QC revealed an unusual H-bond (hydrogen-bond) network in the active site, formed by several highly conserved residues (Ser160, Glu201, Asp248, Asp 305 and His319), within which Glu201 and Asp248 were found to bind to substrate. In the present study we combined steady-state enzyme kinetic and X-ray structural analyses of 11 single-mutation human QCs to investigate the roles of the H-bond network in catalysis. Our results showed that disrupting one or both of the central H-bonds, i.e., Glu201 ?Asp305 and Asp248 ?Asp305, reduced the steady-state catalysis dramatically. The roles of these two COOH?COOH bonds on catalysis could be partly replaced by COOH?water bonds, but not by COOH?CONH2 bonds, reminiscent of the low-barrier Asp?Asp H-bond in the active site of pepsin-like aspartic peptidases. Mutations on Asp305, a residue located at the centre of the H-bond network, raised the Km value of the enzyme by 4.4-19-fold, but decreased the kcat value by 79-2842-fold, indicating that Asp305 primarily plays a catalytic role. In addition, results from mutational studies on Ser160 and His319 suggest that these two residues might help to stabilize the conformations of Asp248 and Asp305 respectively. These data allow us to propose an essential proton transfer between Glu201, Asp305 and Asp248 during the catalysis by animal QCs. ? The Authors.
Subjects
Glutaminyl cyclase (glutaminyl-peptide cyclotransferase, QC); Hydrogen-bond network; Proton transfer; Pyroglutamate (pGlu); Site-directed mutagenesis; Synchrotron; X-ray crystallography
SDGs
Other Subjects
Glutaminyl cyclase (glutaminyl-peptide cyclotransferase, QC); Hydrogen-bond networks; Pyroglutamate (pGlu); Site-directed mutagenesis; Catalysis; Hydrogen bonds; Mutagenesis; Pathology; Peptides; Proton transfer; Synchrotron radiation; X ray crystallography; Enzymes; aspartic acid; carboxyl group; enzyme; glutamic acid; glutaminyl cyclase; histone; peptidase; serine; unclassified drug; water; article; catalysis; controlled study; enzyme active site; enzyme kinetics; enzyme structure; human; hydrogen bond; hydrophilicity; mutation; mutational analysis; nonhuman; nucleotide sequence; priority journal; protein folding; protein stability; proton transport; steady state; Aminoacyltransferases; Animals; Catalysis; Catalytic Domain; Cloning, Molecular; Crystallography, X-Ray; Humans; Hydrogen Bonding; Kinetics; Mutagenesis, Site-Directed; Protein Conformation; Animalia
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