title: Proton inventories constitute reliable tools in investigating enzymatic mechanisms: application on a novel thermo-stable extracellular protease from a halo-alkalophilic Bacillus sp.
abstract: A novel protease designated protease-A-17N-1, was purified from the halo-alkalophilic Bacillus sp. 17N-1, and found active in media containing dithiothreitol and EDTAK(2). This enzyme maintained significant activity from pH 6.00 to 9.00, showed optimum k(cat)/K(m) value at pH 7.50 and 33 degrees C. It was observed that only specific inhibitors of cysteine proteinases inhibited its activity. The pH-(k(cat)/K(m)) profile of protease-A-17N-1 was described by three pK(a)s in the acid limb, and one in the alkaline limb. Both are more likely due t3o the protonic dissociation of an acidic residue, and the development and subsequent deprotonation of an ion-pair, respectively, in its catalytic site, characteristic for cysteine proteinases. Moreover, both the obtained estimates of rate constant k(1) and the ratio k(2)/k(-1) at 25 degrees C, from the temperature-(k(cat)/K(m)) profile of protease-A-17N-1, were found similar to those estimated from the proton inventories of the same parameter, verifying the reliability of the latter methodology. Besides, the bowed-downward proton inventories of k(cat)/K(m), as well as the large inverse SIE observed for this parameter, in combination with its dependence versus temperature, were showed unambiguously that k(cat)/K(m) = k(1). Such results suggest that the novel enzyme is more likely to be a cysteine proteinase functioning via a general acid-base mechanism.
authors.affiliation: University of Ioannina, Department of Chemistry, Ioannina 45110, Greece.
authors.forename: Leonidas G
authors.initials: LG
authors.lastname: Theodorou
authors.forename: Angelos
authors.initials: A
authors.lastname: Perisynakis
authors.forename: Krystalenia
authors.initials: K
authors.lastname: Valasaki
authors.forename: Constantin
authors.initials: C
authors.lastname: Drainas
authors.forename: Emmanuel M
authors.initials: EM
authors.lastname: Papamichael
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011522:Protons; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011522:Protons; D010447:Peptide Hydrolases