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 .
1. We have studied the denaturation difference spectra of glyceraldehyde-3-phosphate dehydrogenase and lactic dehydrogenase in the 270-300mp and 220-250mp wavelength regions.2. When hydrogen ion concentration is varied in thc range from pH 7 to pH 2, the absorption changes a t 230-232 mp. are very similar to those a t 291-293 mp. with both dehydrogenases. The pH dependence of the optical rotatory parameters b, and [1)2']232 follows a very similar pattern.3. The addition of sodium dodecyl sulfate induces hardly any change in the optical rotatory properties of the two dehydrogenases, while the difference spectra both in the near and in the far ultraviolet region closely resemble the corresponding denaturation difference spectra.ethylene glycol exhibit a maximum in the far ultraviolet region a t about 227 mp. At this wavelength, the extent of perturbation of tryptophan is 6.3 times greater than that of tyrosine.5. Calculations based on the far ultraviolet perturbation of aromatic chromophores suggest that the far ultraviolet denaturation difference spectra of the two dehydrogenases may be attributed to changes in the environment of aromatic chromophores, particularly of tryptophan. The solvent perturbation spectra of aromatic chromophores due to 20When the absorption of native and denatured proteins are compared, difference spectra can be obtained in two wavelength regions, between 270 to 300 mp and 220-250 mp, The denaturation-induced spectral changes in the ncar ultraviolet region are attributed to changes in the environment of tyrosyl and tryptophyl side chains, which are buried in the hydrophobic region of the native protein molecule [1,2]. It is known that the difference in absorption which is measured at 292-293 mp corresponds to changes in the environment of tryptophyl side chains, while the difference measured a t 286-287 mp is due to both tyrosyl and tryptophyl side chains [3-51.The origin of the denaturation difference spectra in the far ultra,violet region, that is between 220 and 250 mp, is not yet clear. The observations of Glazer and Smith [6,7] with polyamino acids and proteins show that spectral changes in the 220-250 mp region may be due to peptide chromophores undergoing a helix-coil transition. On the other hand, Edsall and associates [8][9][10] have found that both the near and the far ultraviolet difference spectra of serum albumin and carbonic anhydrase can be accounted for by changes in the absorption of aromatic chromophores.Enzymes. Glyceraldehyde-3-phosphate dehydrogenase or ~-glyceraldehyde-3-phosphate: NAD oxidoreductase (phosphorylating) (EC 1.2.1.12); lactic dehydrogenase or L-lactate: NAD oxidoreductase (EC 1.1.1.27).Glyceraldehyde-3-phosphate dehydrogenase and lactic dehydrogenase have been chosen as model proteins for our studies because both proteins are known to possess a considerably ordered structure with a number of buried chromophores. We suggest that a comparison of denaturation difference spectra, perturbation spectra, and optical rotatory dispersion measurements under diffe...
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