Stability of subtilisins and related proteinases (subtilases)

The stability towards thermal and chemical (guanidine hydrochloride, GnHCl) denaturation of six inhibited subtilases (mesentericopeptidase, subtilisins BPN′, Carlsberg and DY, proteinase K and thermitase) has been investigated by kinetic and equilibrium studies. The unfolding processes were monitored by circular dichroic and fluorescence spectroscopy. Experiments in the absence and presence of extraneous calcium in the concentration range 2×10^?3-10^?1 M were performed. The presence of calcium in the weak calcium binding site changes the denaturation drastically. The heat- (or GnHCl-) induced unfolding curves obtained using CD spectroscopy show two independent transitions which seem not to have been resolved before. The presence of Ca²⁺ in the second (third in the case of thermitase) binding site increases the T_m, values by 11-21 °C and the δG_D(H₂O) values obtained from denaturation experiments in GnHCl by 6.7-7.2 kcal/mol when an extraneous Ca²⁺ concentration of 2 × 10^?2 M was used. One interpretation is that the initial step of denaturation in the presence of added calcium is the formation of a partially unfolded intermediate form, retaining a highly ordered structure with 60-85% of the a-helix structure of the native enzyme. This intermediate then unfolds at a temperature considerably higher than that of the same proteinases in the absence of added Ca²⁺. The free energy of stabilization of the intermediates is increased by 1.8-2.8 times in comparison with that for the unfolding reactions of the subtilases with empty Ca2/Ca3 binding sites. A second interpretation is that the two steps in the unfolding curves correspond to enzyme without and with calcium in the weak binding site. Fluorescence experiments confirm the mechanism involving the formation of intermediate states. The results are discussed in relation to the X-ray models of the six subtilases.     

Studies on the carbodiimide-mediated model couplings of Z-Pro-Leu-OH with benzoaza-15-crown-5

A series of model DCC-mediated couplings of Z-Pro-D-Leu-OH with 2,3-benzo-10-aza-1,4,7,13-tetraoxacyclopentadeca-2-ene were studied by high-performance liquid chromatography. It was found that racemization during preparation of N-(Z-Pro-Leu-)benzoaza-15-crown-5 is promoted by HOBT, an additive usually reducing this process.     

Photoreactivity of histidyl residues in subtilisins Novo and DY. Photooxidation of subtilisins

Subtilisins Novo and DY were photoinactivated in the presence of methylene blue according to first order kinetics. The competitive inhibitor Nα-benzoyl-L-arginine protected significantly against inactivation. Under the conditions employed in this study a selective photooxidation of the active site histidine 64 was achieved. Rate constants of 0.32 times 10^-2, s^-1 and 0.35 times 10^-2, s^-1, were calculated for the Novo enzyme and subtilisin DY, respectively. Apparent pKa values of the catalytically important imidazole group of 7.0 ± 0.1 (s. Novo) and 7.1 ± 0.1 (s. DY) were directly determined. The histidyl residues in the two proteases, except the active site histidine, which is the first target of photooxidation, are “buried” in the interior of the protein globule. Conformational studies suggested that the photoreactive histidine is not involved in the stabilization of the protein conformation.     

FLUORESCENCE PROPERTIES OF NATIVE AND CHEMICALLY MODIFIED MESENTERICOPEPTIDASE

Studies on the fluorescence properties of native mesentericopeptidase as a function of the temperature and/or in the presence of either neutral or ionic fluorescence quenchers demonstrate that the intrinsic emission of this protein is dominated by a partially exposed tryptophyl residue, which is probably located in a site of high dielectric constant containing positively charged amino acid side chains. One largely exposed tryptophan contributes about 14% of the total emission, whereas one deeply buried tryptophan is virtually non-fluorescent. The conversion of the active site serine to cysteine and the insertion of either one phenylmethanesulfonyl or one dansyl substituent into the active site induce only subtle differences in the conformational properties with respect to the native protein; in particular, the mutual distances and orientation between the 13 tyrosyl and 3 tryptophyl residues are unaffected, as shown by singletsinglet energy transfer experiments.     

Spectroscopic studies on proteinase K and subtilisin DY

Circular dichroic spectroscopy has been used to study the effect of pH, guanidinium hydrochloride concentration and temperature on the conformation of the fungal subtilisin-like proteinase K and the bacterial DY. The ellipticity of the bands in the far ultraviolet region remains almost unchanged in the pH range 3.0-11.0 (PMS-proteinase K) and 5.0-10.0 (PMS-subtilisin DY). The same ranges of pH stability were determined from the pH dependence of the near ultraviolet dichroic spectra. Hence the changes in the tertiary and secondary structure occur in parallel. Proteinase K is considerably more stable at acidic and somewhat more stable at alkaline pH than subtilisin DY. At neutral pH proteinase K is more resistant to denaturation by guanidinium hydrochloride than is subtilisin DY. The midpoints of the denaturation curves were 6.2 M and 3.2 M guanidinium, respectively. The thermal unfolding of proteinase K occurred at a higher temperature than for subtilisin DY, the transition midpoints being 65° and 48°, respectively. Thus proteinase K is overall a much more robust molecule than subtilisin DY, showing greater resistance to all three forms of denaturation. The differences in the stability of the two proteinases can be partly explained by differences in their calcium binding sites.     

pH DEPENDENCE OF THE CIRCULAR DICHROIC BANDS OF PHENYLMETHANESULFONYL-MESENTERICOPEPTIDASE

The effect of pH on the circular dichroism spectra of phenylmethanesulfonyl-mesentericopeptidase (peptidyl peptide hydrolase, EC 3.4.21) was studied. The ellipticity of the bands below 250 nm, which reflects the backbone conformation of the protein molecule, remains almost unchanged in the pH range 6.2–10.4. However, below pH 6.2 and above pH 10.4 a conformational transition occurs. The pH-dependent changes above 250 nm were also studied. The titration of the CD band at 296 nm reflects the ionization of the “exposed” tyrosines, which phenolic groups are fully accessible to the solvent. An apparent pK of 9.9 is calculated from the titration curve. It is concluded that ionization of the tyrosyl residues with normal pK's is complete before conformational changes in the protein molecule occur.