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Journal of Biochemistry Advance Access originally published online on July 23, 2007
Journal of Biochemistry 2007 142(2):293-300; doi:10.1093/jb/mvm132
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© 2007 The Japanese Biochemical Society.

Proton Inventories Constitute Reliable Tools in Investigating Enzymatic Mechanisms: Application on a Novel Thermo-stable Extracellular Protease from a Halo-Alkalophilic Bacillus sp.

Leonidas G. Theodorou, Angelos Perisynakis, Krystalenia Valasaki, Constantin Drainas and Emmanuel M. Papamichael*

University of Ioannina, Department of Chemistry, Ioannina 45110, Greece

*To whom correspondence should be addressed. Tel: +30 2651 098395, Fax: +30 2651 047832, E-mail: epapamic{at}cc.uoi.gr

Received May 9, 2007; Accepted June 13, 2007


  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 EDTAK2. This enzyme maintained significant activity from pH 6.00 to 9.00, showed optimum kcat/Km value at pH 7.50 and 33°C. It was observed that only specific inhibitors of cysteine proteinases inhibited its activity. The pH-(kcat/Km) profile of protease-A-17N-1 was described by three pKas 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 k1 and the ratio k2/k–1 at 25°C, from the temperature-(kcat/Km) 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 kcat/Km, as well as the large inverse SIE observed for this parameter, in combination with its dependence versus temperature, were showed unambiguously that kcat/Km = k1. Such results suggest that the novel enzyme is more likely to be a cysteine proteinase functioning via a general acid-base mechanism.

Key Words: cysteine proteinases, enzyme kinetics, enzyme mechanisms, halo-alkalophiles, proton inventories

Abbreviations: DMSO, dimethyl sulphoxide; E-64, 1-[[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]amino]-4-guanidinobutane; ONp, p-nitrophenyl; PI, proton inventory and/or inventories; PCMB, 4-hydroxy-sodium-mercury-benzoate; pNA, p-nitroanilide; SIE, solvent isotope effect; Suc, succinyl


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