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A model to explain the pH-dependent specificity of cathepsin B-catalysed hydrolyses.

ABSTRACT: 1. Three synthetic substrates of cathepsin B (EC 3.4.22.1) with various amino acid residues at the P2 position (Cbz-Phe-Arg-NH-Mec, Cbz-Arg-Arg-NH-Mec and Cbz-Cit-Arg-NH-Mec, where Cbz represents benzyloxycarbonyl and NH-Mec represents 4-methylcoumarin-7-ylamide) were used to investigate the pH-dependency of cathepsin B-catalysed hydrolyses and to obtain information on the nature of enzyme-substrate interactions. 2. Non-linear-regression analysis of pH-activity profiles for these substrates indicates that at least four ionizable groups on cathepsin B with pKa values of 3.3, 4.55, 5.46 and greater than 7.3 can affect the rate of substrate hydrolysis. 3. Ionization of the residue with a pKa of 5.46 has a strong effect on activity towards the substrate with an arginine in P2 (8.4-fold increase in activity) but has only a moderate effect on the rate of hydrolysis with Cbz-Cit-Arg-NH-Mec (2.3-fold increase in activity) and virtually no effect with Cbz-Phe-Arg-NH-Mec. The kinetic data are consistent with this group being an acid residue with a side chain able to interact with the side chains of an arginine or a citrulline in the P2 position of a substrate. Amino acid sequence alignment and model building with the related enzyme papain (EC 3.4.22.2) suggest that Glu-245 of cathepsin B is a likely candidate. The relative importance of electrostatic and hydrophobic interactions in the S2 subsite of cathepsin B is discussed. 4. For all three substrates, activity appears after ionization of a group with a pKa of 3.3, believed to be the active-site Cys-29 of cathepsin B. The identity of the groups with pKa values of 4.55 and greater than 7.3 remains unknown.

SUBMITTER: Khouri HE

PROVIDER: S-EPMC1150117 | biostudies-other | 1991 May

REPOSITORIES: biostudies-other

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Project description:BackgroundDextransucrases are extracellular enzymes, which catalyze the formation of α-1→6-linked glucose polymers from sucrose. These enzymes are exclusively expressed by lactic acid bacteria, which commonly acidify the extracellular environment due to their physiology. Dextransucrases are thus confronted with steadily changing reaction conditions in regards to the environmental pH, which can further affect the amount of released dextransucrases. In this work, we studied the effect of the environmental pH on the release, the productivity and the product specificity of the dextransucrase expressed by Lactobacillus (L.) hordei TMW 1.1822. Dextransucrases were recovered as crude extracts at pH 3.5-pH 6.5 and then again used to produce dextrans at these pH values. The respectively produced dextran amounts and sizes were determined and the obtained results finally systematically correlated.ResultsMaximum dextran amounts were produced at pH 4.0 and pH 4.5, while the productivity of the dextransucrases significantly decreased at pH 3.5 and pH 6.5. The distribution of dextran amounts produced at different pH most likely reflects the pH dependent activity of the dextransucrases released by L. hordei, since different transglycosylation rates were determined at different pH using the same dextransucrase amounts. Moreover, similar hydrolysis activities were detected at all tested conditions despite significant losses of transglycosylation activities indicating initial hydrolysis prior to transglycosylation reactions. The molar masses and rms radii of dextrans increased up to pH 5.5 independently of the stability of the enzyme. The gelling properties of dextrans produced at pH 4.0 and pH 5.5 were different.ConclusionsThe presented methodological approach allows the controlled production of dextrans with varying properties and could be transferred and adapted to other microbes for systematic studies on the release and functionality of native sucrases or other extracellular enzymes.

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A model to explain the pH-dependent specificity of cathepsin B-catalysed hydrolyses.

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