1. The effect of maltose was studied in porcine pancreatic amylase. At neutral p H 101, (29 mM) maltose produced with amylase a difference spectrum characteristic of the perturbation of tryptophan. The molar absorption Werence a t the maximum wavelength was AE~,,, = 1200.2. The difference spectrum appeared to be specific for maltose. Perturbation difference spectra measurements in 20°/, polyethylene glycol indicated that one tryptophyl side chain per mol amylase was involved in the interaction with maltose.3. The dissociation constant of the amylase * maltose complex calculated from the concentration dependence of the absorption difference a t 290 nm was Ks = 13 mM. Maltose inhibited amylase activity competitively and an inhibition constant of Ki = 25 mM was obtained, a similar value to that found spectrophotometrically. It is assumed that the tryptophyl side chain interacting with maltose may be involved in the binding of substrate by pancreatic amylase.It has been known for a long time that n-amylases of various origins split 0~-1,4-glucosidic bonds in a random manner which finally produces mainly maltose from starch, glycogen or dextrins (cf. Fischer and Stein [l]). So far, the action of the enzyme has been studied by analyzing the various products of the enzymatic process. Practically no information is available about the nature of side chains of pancreatic amylase which participate in the binding and hydrolysis of polysaccharide substrate. It was found with Bacillus subtilis amylase that oxidation of one tryptophyl side chain completely inactivated the enzyme [2], but did not alter its immunological properties [3]. Modification of several tyrosyl side chains in the bacterial enzyme with tetranitromethane or acetylimidazole also resulted in the loss of enzyme activityThe enzyme substrate interaction can be studied in some cases also by means of spectrophotometry, when the binding of the substrate or product to a chromophoric group of an enzyme causes changes in its environment. It was shown by Hayashi et al. [5] that the binding of substrate analogues to lysozyme produced difference spectra, which were characteristic of the perturbation of one tryptophyl side chain per mol enzyme [6]. A related phenomenon was observed by Benmouyal and Townbridge [7] with trypsin and chymotrypsin .
The kinetic behaviour of human thrombin has been studied with 26 tripeptidyl-p-nitroanilide substrates protected at the N terminus and with 9 unprotected ones. By the regression analysis of experimentally determined I/&,, k,,, and /cCat/Km values the individual contribution of each side chain of the various substrates to the kinetic parameters was calculated.The contributions to the kinetic parameters of the best substrates provide information about the structure of the binding site. The interaction of subsites S1 and PI, which determines primary specificity, proved to be marginal on the basis of contribution values, though it depends upon this contact whether the substrate is hydrolyzed at all. At subsite Sz proline appeared to be favourable. Subsite S3 plays an important role in efficiency. The best parameters were obtained here with the D configurations of bulky amino acid residues. The aromatic protecting groups applied did not improve the properties of substrates. BzDPhe-Pro-Arg-Nan was predicted by calculation to be better than the protected substrates assayed. The compound was synthesized and tested. Its experimentally determined l/K,,,, 55.1 mM-', was in good agreement with 50.9 mM-' found by calculation.Several serine proteases, e.g. thrombin, trypsin, plasmin and factor Xa, hydrolyze peptide bonds at the carboxylic group of basic amino acids. Their sensitivities to substrate specificity are, however, different. Among them thrombin is the most specific which, apart from a few exceptions, splits next to arginine [1,2] [9] and growth hormones [lo]. According to Muszbek [ I l l in actin it also hydrolyzes next to lysine, whereas hydrolysis does not occur next to ornithine [12].The detailed structure of the substrate-binding sites of thrombin is not yet known. The investigations on the specificity and substrate-binding sites have so far been carried out, in addition to fibrinogen, with peptide derivatives analogous to fibrinogen sequences [13,14] [2,16] and similar hydrophobic side chains, whereas results concerning the S3 substrate-binding subsite are contradictory. Scheraga holds that S3 is rather narrow; it is conceivable, though, that more than one binding domain is operative against long peptides [12].In an attempt to study the substrate-binding sites of thrombin we applied a mathematical method [19], by the aid of which, through the regression analysis of the kinetic constants measured for various tripeptidyl-p-nitroanilide substrates, the interaction between substrate side chains and the
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.