A conserved hydrogen-bond network in the catalytic centre of animal glutaminyl cyclases is critical for catalysis

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 K_m value of the enzyme by 4.4-19-fold, but decreased the k_cat 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.