The catalytic mechanism of archaeal inorganic pyrophosphatase revealed by X-ray and neutron crystallography

A structure-based model for the catalytic cycle of archaeal inorganic pyrophosphatase (IPPase) from the sulfur-reducing hyperthermophilic archaeon Thermococcus thioreducens has been proposed providing new insight into a mechanism for proton transfer-mediated, metal-assisted enzyme catalysis. Eight high-resolution X-ray crystallographic structures of the enzyme have been determined in complex with the substrate, product, and in the presence of different metal cofactors and reactive analogues. As a result, structural snapshots of the catalytic site revealed important catalytic events that could not have been obtained otherwise. Moreover, a method for growing large volume protein crystals (>1 mm3) suitable for neutron diffraction studies has been developed through an advancement of the capillary counter-diffusion crystallization technique in restricted geometry. Large volume crystals of IPPase were obtained and used for neutron crystallographic structure determination of the enzyme solely bound to Ca2+ at 2.50A resolution. The IPPase neutron crystallographic structure provided further structural information at the level of water orientation and the definitive location of hydrogen atoms in the catalytic site. The archaeal enzyme was also observed to exhibit active site rearrangements and activities closely resembling those described in other investigations conducted on bacterial and eukaryotic IPPases. In this investigation, it was hypothesized that Asp68 in T. thioreducens IPPase (equivalent to Asp67 and Asp 117 in E. coli and S. cerevisiae respectively) was the most likely principal proton acceptor for the deprotonation of a water molecule that bridges two metals, resulting in the formation of an attacking hydroxide nucleophile toward the hydrolysis of the pyrophosphate (PPi) substrate. This hypothesis was tested by measuring the enzymatic activity of an Asp68Asn mutant construct of T. thioreducens IPPase and comparing it with the activity of the unaltered recombinant enzyme. Consequently, it was determined that the mutant enzyme retained significant activity (∼10-15%) towards PP i hydrolysis. This finding led to the proposal of an alternative pathway for nucleophile activation in T. thioreducens IPPase that is independent of Asp68. In the alternate pathway, nucleophile activation is accomplished by transferring the proton along a chain of water molecules to the bulk solvent. Thus, the X-ray and neutron crystallographic models coupled to supporting biochemical data allowed, for the first time, a structure-based mechanism for IPPase catalysis from an archaeal system to be proposed.