Investigation of the Active Center of Porcine‐Pancreatic Amylase

Abstract

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 Mechanism of Porcine Pancreatic α‐Amylase

“…Difference spectra of the maltose-porcine enzyme complex have been obtained by Elodi and Mora (1972). A similar experiment was performed here using acarbose as a Iigand.…”

Relationship between ?-amylase degradation and the structure and physicochemical properties of legume starches

“…These authors have shovm that only chain to the right diffuses away after the initial cleavage and the remaining chain to the left diffuses to fill the open binding subsites to give maltose (G2), maltotriose (G3) and maltotetraose (G4) as products in a multiple attack mechanism. The products of hydrolysis, particularly G2 and G3, are known to have an inhibitory effect on the action of a-amylase in vitro (Robyt and French, 1970;Elodi et al, 1972; 97 Leloup et al, 1991). G2 and G3 have been shown to bind strongly to PPA, thereby impeding their adsorption onto crystalline spherulites of short chain amylose (Leloup et al, 1991).…”

Some Characteristics of an Alpha-amylase Inhibitor fromPhaseolus vulgaris(Cultivar Great Northern) Seeds

“…8) The molarity of the IX-amylase solution was calculated from the absorbance at 280 nm, on the basis of a molecular mass of 52 kDa and A = 24.0 (1 %, 280 nm) by the method of Elodi et al 9 ) Amylase and amylase inhibitory activities were measured essentially by the method of Dahlqvist 10 ) using soluble starch as a substrate. One unit of inhibitor was defined as the amount of inhibitor that gave 50% inhibition of the activity of 1 jlg of purified porcine pancreatic amylase.…”