Involvement of the NH2-terminal region of oryzacystatin-I in cysteine proteinase inhibition

Abstract

Dihydroorotase is the central domain of trifunctional L-dihydroorotate synthetase which also contains carbamyl phosphate synthetase at the N-terminus and aspartate transcarbamylase at the C-terminus. The cDNA, corresponding to the active dihydroorotase domain as isolated after digestion of dihydroorotate synthetase with elastase, has been sub-cloned into the expression vector pCW12 which was then used to transform Escherichia coli SØ1263 pyrC⁻ lacking dihydroorotase activity. However, induction of this recomhinant strain with IPTG produced large amounts of the dihydroorotase domain which were completely inactive. A number of cDNAs were expressed which were longer on the C-terminal side; all cDNAs expressed active dihydroorotase domain down to a minimal extension of 12 ammo adds (-Val- Pro-Pro-Gly-Tyr-GIy-Gm-Asp-Val-Arg-Lys-Trp) into the bridge region between the dihydroorotase and aspartate transcarbamylase domains. Part of this dodecapeptide may form an amphipathk helix which in some way constrains the isolated, recombinant dihydroorotase domain to an active conformation. The recombinant hamster dihydroorotase purified from a cell-free extract of E.coli in four steps has a turnover number of 297 mol/min/(mol domain) for the conversion of L-dihydroorotate back to N-carbamyl-Laspartate with K₈ = 8.7 ± 1.5 μM for L-dihydroorotate, a subunit molecular weight of 39 008 determined from the sequence and 37 900 ± 400 when subjected to SDS–PAGE, and an isoelectric point of 5.7. Ultracentrifugal analysis of the recombinant domain showed a single species of s_20,w = 4.1 S and a single molecular species of M_r = 76 000 corresponding to a dimer.