Investigations into the enzymology and biotechnology of the hyperthermophilic carboxypeptidase (PfuCP) from the archaeon Pyrococcus furiosus

A novel metallocarboxypeptidase (PfuCP) from the hyperthermophilic archaeon Pyrococcus furiosus was purified and characterized to investigate its dependence on metal ion cofactors and to evaluate its suitability as a biotechnological tool for protein sequencing at elevated temperatures.; The crystal structure reveals a dimer of primarily α-helical subunits that bears no resemblance to the α/β-hydrolase morphology of typical carboxypeptidases and which defines a new family of HEXXH metalloproteases (M32) based on primary sequence alignments. A deep active site groove appears to function not only in size-selection of substrates but also in modulating the activity and substrate affinity through complicated allosteric effects involving ambient ligands which may play a role in regulatory metabolism.; Two forms of the enzyme were observed; one which retains stabilizing metal(s) that confer structural thermostability and a remarkable retention of activity to the dimer, and another demetallated form which has lost stability with regards to both dimeric integrity and activity. Difficulties in expressing a properly folded recombinant necessitated refolding of the expressed clone from inclusion bodies and further suggest that in vivo the stabilizing metal(s) may participate in folding a metastable enzyme.; The apparent paradox of activation by only Co2+ and not Zn2+ is resolved into two issues, uncompetitive inhibition by the latter as seen in steady-state kinetic experiments, and intrinsic, electronic aspects of a catalytic Co2+. Several explanations are proposed for the intrinsic rate enhancement of Co2+ over Zn2+ including the ability of Co2+ to modulate the potential energy surface for both reactants and transition states by virtue of its greater mobility within the protein framework.; The broad amino acid specificity and rapid digestion by PfuCP in peptide sequencing trials show promise, and high-temperature protein sequencing has now been demonstrated for the first time.