The Enzyme-substrate Complex in a Muramidase Catalyzed Reaction. I. Difference Spectrum of Complex. By KATSUYA HAYASHI, TAIJI IMOTO and MASARU FUNATSU

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1 The Journal of Biochemistry, Vol. 54, No. 5, 1963 The Enzyme-substrate Complex in a Muramidase Catalyzed Reaction I. Difference Spectrum of Complex By KATSUYA HAYASHI, TAIJI IMOTO and MASARU FUNATSU (From Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka) The concept that an enzyme molecule which is actively catalyzing a reaction may have different characteristics from one which is not in contact with substrate, has been proved sometimes for the whole structure, sometimes for the active center, by many observations. This concept of the flexibility of the enzyme molecule was called the " In duced fit theory". Recentry, Y a g i and O z a w a (1) have stated that a conformation change of the n- amino acid oxidase molecule was observed when it forms the Michaelis complex with the substrate or a complex with a competi tive inhibitor such as benzoic acid. Further more, many authors (2, 3) have reported in detail that the intermediate, acyl chymotry psin, in the chymotrypsin [EC cataly zed reaction of p-nitro phenyl acyl ester, ex hibited difference in its spectroscopic charac ter from the untreated enzyme. We have been studying the behavior of the muramidase [EC , N-acetylmura mide glucanohydrolase] molecule in a re action mixture by comparing it with the enzyme in a buffer solution with no substrate present. Changes in the shape and intensity of the ultraviolet absorption spectrum of muramidase were observed to occur owing to the formation of an enzyme-substrate complex, when a soluble chitin derivative, glycol chitin, was used as the substrate. To study these changes quantitatively, the tech nique of difference spectrophotometry has great advantages, and allows the determi nation of some features of the protein structure surrounding the chromophores in the peptide chain. The difference spectrum of muramidase in the reaction mixture (Received for publication, December 10, 1962) 381 measured against untreated muramidase as a reference, exhibited a specific pattern in which three peaks could be read. The difference intensities at mp (D293.5) are pro portional to the enzyme activity measured under various circumstances. This article deals mainly with the be haviour of muramidase in the reaction mixture, as summarized from the difference spectra data. EXPERIMENTAL Muramidase-The muramidase preparation was derived from egg white by the direct crystallization method, recrystallized at least five times and lyo philized. The preparation showed a single peak on CMC and IR-50 column chromatograms. Glycol chitin-glycol chitin was synthesized from purified chitin by S e n z y u's method (4) with only minor modification. After washing three times with 80%o ethyl alcohol, the glycol chitin was disolved in a small amount of water, and the ph was brought to 7 by adding dilute hydrochloric acid. After dia lyzing against water for three days and filtering on paper, the filtrate was lyophilized and stored. The molecular weight of the glycol chitin thus obtained was found to be of order of 90, ,000 by vis cosimetry. The ultraviolet absorption of glycol chitin at a concentration of 0.33 mg./ml. which was used in most of the experiments reported below, was low and very flat in the entire ultraviolet region as shown in Fig. 1. No change in the absorbancy of glycol chitin was found during its degradation by muramidase. It is possible, therefore, to ignore any changes in the ultraviolet absorption of the substrate in these ex periments. Activity Assay by Viscosimetry-The measurement of the enzymatic activity of muramidase by viscosi metry was carried out by the method described by Hamaguchi and Funatsu (5). The reaction

382 K. HAYASHZ, T. IMOTO and M. FUNATSU acetate buffer, ph 4,0, and muramidase was also dissolved in these solutions so that the final concen tration of muramidase becomes 5mg./ml.

2 382 K. HAYASHZ, T. IMOTO and M. FUNATSU acetate buffer, ph 4,0, and muramidase was also dissolved in these solutions so that the final concen tration of muramidase becomes 5mg./ml. The re action mixtures were allowed to stand at room tem perature for 30 minutes to complete the reaction. The extent of decomposition of tryptophan residues was measured spectroscopically by following the decrease in optical densities at 278 m/p and using W i t k o p's factor, 1.3, by setting the molecular ex tinction coefficient of tryptophan equal to 5,500 (6). WAVELENGTH (mu) Fro. 1. Ultraviolet spectrum of glycol chitin. Concentration of glycol chitin : 0.33 mg./ml. in 0.1 M phosphate, ph 5.6. mixture consisted of 10 ml. of 0.08% glycol chitin and 0.2 ml. of 0.024% muramidase in 0.1 M phos phate buffer, ph 5.6. The specific viscosity of this solution decreases with time, owing to the degradation of the glycol chitin. The specific viscosity of the solution, expressed as in terms of a percentage of the specific viscosity at zero time is plotted against the time, and the value at three minutes of incubation was taken for the activity determination. When samples of chemically modified muramidase were used, theiractivity was determined from the standard curve for untreated muramidase. The ratio of the concentration determined in this way, to the known concentration, was called as the ` relative activity '. Activity Assay by Measuring Reducing Power-To 0.2% glycol chitin in 0.1 M phosphate buffer, ph 5.6, was added a tenth volume of 0.05% muramidase solution in the same buffer. The reaction mixture was then kept at 50 C for four hours and the re ducing power was analyzed by S o m o g y is method (titration with sodium thiosulfate), and calculated as an amount of N-acetyl glucosamine in mg./ml. Difference Spectrum Measurement-All difference spectra in the ultraviolet region ( m!i) were recorded using a Beckman DU spectrophotometer with quartz cell of 1 cm. light path. The reference cell contained a solution of muramidase, and the experimental cell contained a solution of muramidase, at the same concentration, plus substrate. The ab sorption of the added reagent was recorded separately and a correction was made to the difference spectrum by subtracting its absorbancy at each wave length. All runs were done at 18 C, and in the case in which the added material is substrate, the difference spectrum was recorded within the range of minutes after substrate was added to enzyme solution. N-bromosuccinimide Treatment-N-bromosuccinimide was dissolved at various concentrations in 0.1 M RESULTS 1. The Difference Spectrum of Muramidase with Glycol Chitin-Muramidase in the presence of the substrate, glycol chitin, showed a dif ference spectrum which has peaks at 275, 285 and my as shown in Fig. 2. The peak appearing at my is the largest in an ordinary solvent and the most characteristic. The difference in the optical density at WAVELENGTH (MP) FIG. 2. Difference spectrum of muramidase with glycol chitin. Concentration of muramidase: 0.33 mg./ml., concentration of glycol chitin : 0.33 mg. /ml., in 0.1 M phosphate, ph 5.6. Spectrum was recorded at 18 C within 30 minutes after glycol chitin was added to muramidase solution. my is denoted by ƒ D The dependence of ƒ D293,5 on the glycol chitin concentration with a fixed amount of muramidase is shown in Fig. 3 where a plot of ƒ D293,5 against the concentration of glycol chitin in the mixture is shown. AD,,,., reached a maximum when the concentration of glycol chitin nearly equalled the concentration of the muramidase in the mixture. Maximum values of ƒ D293.5 and As were found to be and respectively, for muramidase at a concentration of 0.33

3 E-S Complex of Muramidase 383 CONCENTRATION OF GLYCOL CHITIN (mg./mi.). FIG. 3. Change in difference intensity with glycol chitin concentration. Concentration of ' muramidase : 0.33 mg. /ml. O : intensity at mp, 0 : intensity at 285 rip. mg./ml., and these values are fairly large for an optical density difference in neutral so lution. 2. Verification that the Difference Spectrum Originates from Specific Interaction of Enzyme with Substratc- Several recordings of difference spectra were made on free tyrosine and try ptophan solutions with addition of glycol chitin, since the origin of difference spectra is in changes of the environment of the chromophores, the tyrosine and tryptophan residues, in the muramidase molecule. As shown in Fig. 4, nothing was observed in these systems. This shows that the unique difference spectrum appearing in the mura midase-substrate mixture was not caused by a non-specific interaction between glycol chitin and tryptophan or tyrosine residues. Another run was done on an ovalbumin-glycol chitin mixture which, of course, does not form an enzyme-substrate complex. As expected, no difference spectrum was observed. Glycol chitin at such a low concentration as 0.33 mg./ml. would not be expected to affect the protein spectrum appreciably, by a solvent effect, and, at least with ovalbumin, this ex pectation is borne out. 3. Effect of Other Reagents-Several re agents which are closely related in their chemical structure to the substrate were mixed into muramidase solution to see whether they affected the spectrum of the enzyme. The following were selected; glycol chitosan, glycol cellulose, soluble starch, dextran, maltose, glucose, glucosamine hydrochloride, N-acetyl glucosamine, octacetyl chitobiose and N, N'-diacetyl chitobiose. Nothing was observed except with the N, N'-diacetyl chito biose. Muramidase solutions containing N, N'-diacetyl chitobiose showed a remarkable difference spectrum in which both the shape and the locations of peaks are quite similar to those obtained with glycol chitin as shown in Fig. 5. It is obvious that the difference spectrum appears only in the case in which an N-acetyl glucosamine polymer, including the dimer, was mixed with the muramidase solution. WAVELENGTH(my) FIG. 4. Difference spectra of tyrosine, try ptophan and ovalbumin with glycol chitin. Concentration of glycol chitin : 0.33 mg./ml., tyrosine : 0.09 mg./ml., tryptophan : 0.03 mg./ ml., ovalbumin : 1.3 mg./ml., buffer: 0.1 M phosphate, ph 5.6. WAVELENGTH (mt) FIG. 5. Difference spectra of muramidase with various reagents. Concentration of murami dase : 0.33 mg./ml. Curve I : N, N'-diacetyl chito biose, concentration: 0.33 mg./ml. Curve II : partially hydrolyzed product, concentration : 0.33 mg./ml. Curve III: N-acetyl glucosamine, concentration: 0.5 mg./ml.

4 384 K. HAYASHI, T. I EoTo and M. FUNATSU 4. Proportionality of Difference Intensity to Activity. a) ph Dependence-Fig. 6 was drawn by plotting AD,,,., observed at various ph values. The curve thus obtained was similar to that for ph-dependence of the activity obtained by the reducing power method, the maximum of both curves being equally at ph 5.5. It differes slightly from the activity curve ob tained by viscosimetry. b) Changes in Difference Intensity and Activity with Changing Urea Concentration-As shown in CONCENTRATION OF UREA ( 41 ) FIG. 7. Effect of concentration of urea on activity and difference intensity. : ƒ ƒ D393.5, C : relative activity measured by viscosimetry. FIG. 6. ph-dependence of activity and dif ference intensity. Concentration of both solutes, muramidase and glycol chitin, is 0.33 mg./ml. : ƒ D293.5, X : relative activity measured by vis cosimetry, 0 : relative activity by reducing power method. Relative activity at ph 4.5 (for viscosi metry) or ph 5.5 (for reducing power method) was taken to be 100. NBS MOLE/MOLE OF MURAMIOASE F.o. 8. Relation between mole of oxidized tryptophan residue in muramidase molecule and NBS amount added. Mole of oxidized trypto phan residue was calculated according to Wi tkop (6). Fig. 7, AD293.5 decreases with increasing urea concentration. This decrease is duplicated by the decrease in the activity, measured by viscosimetry, (7) under the same conditions. In this case, an equal concentration of urea was in the reference cell so that the ab sorption of the urea would be compensated. c) Difference Intensity and Activity of NBS Treated Muramidase-The number of trypto phan residues destroyed by N-bromosuccini mide, NBS, treatment could be found as shown in Fig. 8. ƒ D293.5 of the modified muramidase whose tryptophan residues were destroyed to various extents, were recorded with addition of glycol chitin at ph 5.6. The solid line in Fig. 9 shows the decrease in ƒ D293.5 as a function of the number of trypto phan destroyed and the filled circle the MOLE OF OXI. Try/MOLE OF MURAMIOASE FIG. 9. Difference intensity and activity of NBS treated muramidase. : relative activity measured by viscosimetry, 0 : relative value of AD,293.5.

5 E-S Complex of Muramidase 385 activity measured by the viscosimetry. Both curves are in good agreement. This coin cidence suggests that the activity of mura midase, with glycol chitin as a substrate, can be measured by reading ƒ D293.5 in the neutral region. DISCUSSION 1. The velocity of hydrolysis of glycol chitin catalyzed by muramidase is very small at a low temperature such as 18 C at which all our experiments were carried out. Only 1% of total glucosaminide linkages were hy drolyzed in 30 minutes at this temperature. From this information and the fact that ƒ D293.5 has not shown any time-dependence due to the breakdown of the substrate by hydrolysis, it is reasonable to assume that the enzyme-substrate complex is stabilized by the reduction of the value of k2 by re ducing the temperature. ƒ D293.5 reaches a constant value immediately after adding the substrate to the muramidase solution, and this may mean that kl is large enough and KS is so large that the enzyme-substrate complex can be detected spectroscopically, where k, is the reaction velocity of substrate to complex and k2 is the reaction velocity of complex to product and KS is substrate constant. 2. Although the difference spectrum ap peared in the presence of diacetyl chitobiose, it is doubtful that this compound can be hydrolyzed to the monomer by muramidase. When glycol chitin was allowed to stand at 50 C for four hours with muramidase, the production of reducing groups ceased, even though enough substrate was available, whereas the difference spectrum still re mained. This fact apparently indicates that the hydrolyzed product can also form a complex with muramidase and that it may result in the inhibition of the catalyzing reaction. So it is reasonable to call the complex formed with the hydrolyzed product an enzyme-inhibitor complex. 3. It seems to be quite certain that the origin of the difference spectra is a specific tryptophan residue. We believe that this specific tryptophan residue is probably located at the active center, because both glycol chitin and diacetyl chitobiose, independent of molecular size, can cause the appearance of difference spectrum, and because ƒ D293.5 is directly proportional to the activity. 4. The optimum ph reported by dif ferent investigators so far for muramidase activity is scattered over a considerable range, say ph 3-6, depending on the assay method used. In general, a much higher ph for the optimum action was obtained by the reducing power method, than by the viscosimetric method. The viscosimetric method is affected most by the early stages of the reaction and the reducing power method is a measure of each hydrolytic step, and the optimum ph for ƒ D293.5, therefore, is affected by both of these steps. 5. The decreases in. ƒ D293.5 and activity with NBS treatment were exactly in parallel. Only one tryptophan residue which is ac cessible to NBS seems to be responsible for both the activity and the difference spectrum because a loss of one tryptophan residue led to an 80% loss of both the activity and the difference spectrum. The detection of a difference spectrum in the enzyme-substrate complex in this muramidase catalyzed reaction is probably the first report for any enzyme substrate mixture. The reasons for success in our ex periment are the following ; i) glycol chitin, the substrate, has no absorption in the ultra violet region, ii) the reaction velocity, k2, is very small but k, is fairly large under the conditions used, and consequently the enzyme substrate complex is very stable. To answer the problem of the origin of the difference spectrum or the question as to what is the state of tryptophan residues of muramidase in the reaction mixture, more detailed ex periments must be done for the muramidase catalyzed reaction. Considering the know ledge on this subject reported so far (8-16), some discussion can be allowed. The simplest explanation is that the difference spectrum is caused by a change in electronic state of a tryptophan residue which is near or in the active center of muramidase on the formation

6 386 K. HAYASHI, T. IMOTO and M. FUNATSU of the enzyme-substrate complex. A protein containing tryptophan, which is denatured, generally shows a difference spectrum which is similar to that of the enzyme-substrate complex, but of opposite sign. The blue shift caused by the denaturation has been explained in terms of the change in the environment of tryptophan resulting from the destruction of a hydrophobic region by the denaturant. The difference spectrum of muramidase with glycol chitin is caused by a red shift in the spectrum of the enzyme. Since this is just the opposite of blue shift, new hydrophobic region around the tryptophan residue could be produced by the substrate. This could mean that the muramidase molecule becomes folded into a new conformation when it combines with substrate or that the difference spectrum is caused by the interaction of a tryptophan residue and the substrate. The results reported by F o s s will support this explanation (17). On the other hand, muramidase shows the same difference spectrum in 8 M urea solution as it does in the reaction mixture. This fact indicates also that the difference spectrum with the substrate may be caused by the solvent effect discussed by B i g e l o w and G e s c h w i n d (18). However, this term can be ignored as discussed above. The dif ference spectrum could, therefore, be caused by one of two posibilities; 1) rearrangement in the enzyme mole cule, causing an increase in the hydrophobic environment of one tryptophan residue, 2) the direct binding of a tryptophan residue to the substrate. We can not decide which possibility could be the origin of the difference spectrum at the moment. SUMMARY 1. The difference spectrum of mura midase in the reaction mixture was observed to occur owing to the formation of an enzyme substrate complex. The difference intensities at mu, ƒ D293,5, are proportional to the enzymatic activities measured under various conditions. 2. Free tyrosine and tryptophan solutions with addition of glycol chitin, the substrate for muramidase, did not provoke the dif ference spectrum. An ovalbumin-glycol chitin mixture which does not form an enzyme substrate complex also did not show the difference spectrum. The difference spectrum appeared only in the case in which an N- acetylated glucosamine polymer, including the dimer, was mixed with the muramidase solution. 3. The decreases in ƒ D293.5 and activity with NBS treatment were exactly in parallel. Only one tryptophan residue which is most accessible to NBS seems to be responsible for both the activity and the appearance of difference spectrum. 4. The difference spectrum may be caused by a change in a state of a tryptophan residue which is near or in the active center of muramidase on the formation of the enzymesubstrate complex. And the difference spectrum is caused by a red shift in the spectrum of muramidase. 5. It is assumed that new hydrophobic regions around the tryptophan residue of the muramidase molecule were produced by the substrate on the formation of the enzyme substrate complex. The authors wish to thak Dr. C.C. Bigelow of the University of Alberta for helpful advices and valuable discussions. REFERENCE (1) Yagi, K., and Ozawa, T., Biochim. et Biophys. Acta, 62, 397 (1962) (2) Wootton, J.F., and Hess, G.P., J. Am. Chem. Soc., 84, 440 (1962) (3) Bender, M.L., Schonbaum, G.R., and Hamilton, G.A., J. Polymer Sci., 49, 75 (1961) (4) Senzyu, R., and Okimasu, S., J. Asr. Chem. Soc. Japan (in Japanese), 23, 437 (1950) (5) Hamaguchi, K., and Funatsu, M., J. Biochem., 46, 1695 (1959) (6) Witkop, B., Advances in Protein Chem., 16, 221 (1961) (7) Hamaguchi, K., Rokkaku, K., Funatsu, M., and Hayashi, K., J. Biochem., 48, 351 (1960) (8) Laskowski, M., Leach, S.J., and Scheraga, H.A., J. Am. Chem. Soc., 82, 571 (1960) (9) Williams, E.J., and Foster, J.F., J. Am. Chem.

7 -S Complex of Muramidase 387 Soc., 81, 865 (1959) (10) Wetlaufer, D.B., Edsall, J.T., and Hollingworth, B.R., J. Biol. Chem., 233, 1421 (1958) (11) Yanari, S., and Bovey, F.A., J. Biol. Chem., 235, 2818 (1960) (12) Leach, S.J., and Scheraga, H.A., J. Biol. Chem., 235, 2827 (1960) (13) Donovan, J., and Scheraga, H.A., Biochim. et Biophys. Acta, 29, 455 (1958) (14) Chervenka, C.H., Biochim. et Biophys. Acta, 31, 85 (1959) (15) Nelson, C.A., and Hummel, J.P., J. Biol. Chem., 273, 1576 (1962) (16) Scheraga, H.A., Biochim. et Biophys. Acta, 23, 196 (1957) (17) Foss, J.G., Biochim. et Biophys. Acta, 47, 569 (1961) (18) Bigelow, C.C., and Geschwind, I.I., Compt. rer:d, lab. Carlsberg, 31, 283 (1960)

Hydrolysis of Irradiated Ovalbumin by Pepsin

Hydrolysis of Irradiated Ovalbumin by Pepsin HECTOR A. DIEU and V. DESREUX From the Department of Physical Chemistry, University of Liege, Liege, Belgium ABSTRACT Solid ovalbumin has been irradiated at