ROLE OF THE CHYMOTRYPSIN-LIKE MEMBRANE-ASSOCIATED PROTEINASE FROM TREPONEMA-DENTICOLA ATCC-35405 IN INACTIVATION OF BIOACTIVE PEPTIDES

The ability of washed whole cells of Treponema denticola ATCC 35405 to hydrolyze (inactivate) substance P, bradykinin, and angiotensin I was studied, Substance P was attacked primarily at the Phe-8-Gly-9 bond b y a chymotrypsin-like proteinase (CTLP), at Pro-4-Gln-5 by an endo-act ing prolyl oligopeptidase (POPase), and at Gln-5-Gln-6 by an endopepti dase (FALGPA-peptidase). Bradykinin was cleaved at Phe-5-Ser-6 by the FALGPA-peptidase and at Pro-7-Phe-8 by the POPase, Angiotensin I,vas r apidly converted to angiotensin II by the CTLP, and both angiotensin I and angiotensin II were further hydrolyzed at Pro-7-Phe-8 by the POPa se, All these enzymes were assumed to be cell associated and were easi ly extracted with a mild (0.05 to 0.1%) Triton X-100 treatment, Becaus e it was conceivable that the hydrolysis of substance P at the Phe-8-G ly-9 bond was catalyzed by a CTLP described earlier (V,-J, Uitto, D. G renier, E. C, S. Chan, and B; C, McBride, Infect. Immun, 56:2717-2722, 1988), the enzyme was purified to homogeneity by means of conventiona l fast protein liquid chromatography procedures, For kinetic studies, Phe-8 (4-nitro)-substance P (NSP) (absorption maximum at 309.2 nm, eps ilon = 545 M-l cm-l) was synthesized to replace substance P as a subst rate in kinetic studies. In reversed-phase chromatography, both NSP an d substance P gave identical results with both whole cells and the pur ified enzyme, The CTLP has a mass of 95 kDa, and its activity is sugge sted to be based on an active seryl residue, on an active imidazole gr oup, and on an active carboxyl group but not on metal cations, The enz yme hydrolyzes N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroaniline (SAAP FNA, a typical chymotrypsin substrate) at a high rate and: several pro teins, such as calf thymus histone, human plasma fibrinogen, milk case ins, and gelatin, Among the substrates tested, substance P showed the highest affinity (K-m = 0.22 mM) for the purified enzyme, Depending on conditions, clinically applicable chlorhexidine levels (3.2 mmol/lite r, or 0.2%) strongly activated (up to fourfold) the hydrolysis of SAAP FNA by whole cells and the purified CTLP. The hydrolysis of NSP by who le cells and purified CTLP was slightly inhibited by chlorhexidine. Th e results demonstrated the versatility and the effectiveness of the ou ter membrane of T. denticola in occasioning a rapid breakdown and inac tivation of human bioactive peptides and other peptidolytic catalyses, The tests with whole cells resulted in the accumulation of short pept ides derived from substance P, bradykinin, and the angiotensins, the r esistance of which to further hydrolysis by whole cells deserves addit ional studies.