[Thermitase, a thermostable serine protease of Thermoactinomyces vulgaris: interaction of the active center and the SH-group of the enzyme].

Abstract

Modification of the serine and histidine residue in the active centre of thermitase with diisopropylfluorophosphate (DFP) or L-1-tosylamide-2-phenylethyl chloromethylketon (TPCK), and of the only SH-group of the enzyme, with Hg-compounds causes an activity loss against hydrolysis of 4-nitrophenylacetate. While the modification of cysteine prevents reaction of serine and histidine in the active centre of the enzyme with DFP and TPCK, respectively, the Hg2+- and CF3Hg+-binding to the SH-group after modification of essential amino acid residues in the active centre is retained. To elucidate the interaction of the SH-group with the active centre, the modified products of thermitase were investigated for their thermostability. Ca2+-ions were found to have a stabilizing effect on all the modified products of thermitase, as well as on the native enzyme. Simultaneous modification of the cysteine and serine leads to an increase in thermostability of thermitase, whilst double modification at the cysteine and histidine causes destabilization of the enzyme.