PubMed · 8753436
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M Katoh. 1995. [Chymotrypsin].. https://pubmed.ncbi.nlm.nih.gov/8753436/
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TentaGel, ArgoGel and PEGA resins were evaluated for on-bead biological screening, using a fluorescently-labelled peptide attached to each and assayed for papain activity. Peptide attached to PEGA was cleaved in near quantitative yield at the expected sites, whilst an identical sequence on TentaGel and ArgoGel beads was hydrolysed in very low yields and nonspecifically on ArgoGel. The compatibility of PEGA with enzymes was further demonstrated by the determination of subsite specificities of papain and chymotrypsin using PEGA-bound peptide libraries, which proved to be similar to those observed in free solution.
Resorufin bromoacetate is a substrate that is rapidly hydrolysed by chymotrypsin. The reaction shows a pre-steady-state burst phase that may be observed by stopped flow spectrophotometry if precautions are taken against spontaneous hydrolysis of the substrate. The strongly activating effect that the presence of the bromine atom has on the adjacent carbonyl group is reflected in the relative sizes of the kcat values for resorufin bromoacetate and resorufin acetate (e.g., 740 to 1, at pH 6) and the burst rate constants (e.g., 350 to 1, at pH 7 using 0.1 mM substrate). The pH-dependence of kcat for both substrates shows the involvement of an enzymic group of pKa about 7. With resorufin bromoacetate, a burst and a steady-state rate are still observable at pH 3.0. Unlike the case with aldehyde dehydrogenase, resorufin bromoacetate does not act as an inactivator of chymotrypsin and there is little or no incorporation of covalently-linked label when chymotrypsin and resorufin bromoacetate are incubated together. The different modes of behaviour of the two enzymes are attributable to the 'hard' or 'soft' character of the attacking enzymic nucleophilic groups.
The 64-residue chymotrypsin inhibitor 2 (CI2) folds by a two-state nucleation-condensation mechanism, whereby secondary and tertiary structure coalesce concomitantly in the transition state around Ala 16 in the helical N-cap. Permutation of the SH3-domain of alpha-spectrin apparently shifts its folding nucleus to another region of the protein, suggesting that a protein's transition state may be altered by altering the protein's connectivity. We have characterized the structure of the transition state of a circular and a permuted version of CI2 by a protein engineering study encompassing 11 mutations. Circular CI2 was obtained by the introduction of cysteines at residues 3 and 63 and linking them by disulfide bond formation. Subsequent cyanogen-bromide cleavage of the scissile bond, Met 40-Glu 41, yielded permuted CI2. Circular and permuted CI2 also fold according to a two-state mechanism. Permutation does not affect the folding rate constant, but circularization increases it 7-fold. The transition states of circular and permuted CI2 are essentially unchanged from that of wild-type CI2. Importantly, the folding nucleus around Ala16 is retained. These results complement a previous observation that the transition state for association of two CI2 fragments (residues 1-40 and 41-64, generated by CNBr cleavage) is very similar to the folding transition state of intact CI2. The similarity of rate constants for folding of wild-type and permuted CI2, and their value relative to that for the association of fragments, allows us to estimate the gain in entropy of activation on having the separate fragments linked: 18.3 cal M-1 K-1; i.e. an effective molarity of 10(4) M. The contrast between the retention of the folding nucleus on permutation of CI2 and its change for the SH3-domain of alpha-spectrin probably arises because the latter was cleaved in its folding nucleus whereas cleavage at sites other than 40-41 in CI2 is very destabilizing. Whether or not a folding nucleus can be changed probably depends on the specific protein and its permissivity to permutation.