The dependence of the maximal velocity, Vm with pH for barley malt phosphatase has been studied from pH 4.0 to 5.4. The data show that an ionizable group in the enzyme-substrate complex with a pK2 of 4-4.6 is important for enzymic activity. These results suggest strongly the involvement of a carboxyl group of a glutamic or aspartic acid residue at the active site of this enzyme

Aherne, F. X.

Bennett, G. A.

Berner, D. L.

Burke, M. E.

Busch, N. E.

Carrothers, W.

Chan, K-P.

Conradie, A. R.

Daniels, J. D.

Dunn, F. L.

Feider, M.

Fisher, T. L.

Frobish, L. T.

Ganfield, M. W.

Hallahan, M. E.

Hills, D. C.

Hodgin, L. A.

Howard, J. R.

Hume, D. J.

Huntington, J. L.

Husted, R. R.

Isenhart, J. W.

Johnson, R. J.

Kao, W-H.

Killos, P. J.

Lachica, R. D. F.

Linden, J. C.

Nelson, D. K.

Patnode, S. C.

Qureshi, M. C.

Rambo, R. S.

Richardson, L. F.

Rose, R. J.

Sair, R. A.

Scharffenberg, S.

Shonka, M. L.

Smith, R. L.

Stifel, F. B.

Treadwell, G. E., Jr.

Tsuda, Y.

Wise, J. L., Jr.

University of Northern Iowa

Proceedings of the Iowa Academy of Science Volume 72 Annual Issue Article 19 1965 Identification of a Carboxyl group at the Active Site of Barley MaIt Phosphatase F. X. Aherne Iowa State University G. A. Bennett Iowa State University D. L. Berner Iowa State University M. E. Burke Iowa State University N. E. Busch Iowa State University See next page for additional authors Let us know how access to this document benefits you Copyright ©1965 Iowa Academy of Science, Inc. Follow this and additional works at: https://scholarworks.uni.edu/pias Recommended Citation Aherne, F. X.; Bennett, G. A.; Berner, D. L.; Burke, M. E.; Busch, N. E.; Carrothers, W.; Chan, K-P.; Conradie, A. R.; Daniels, J. D.; Dunn, F. L.; Feider, M.; Frobish, L. T.; Ganfield, M. W.; Hallahan, M. E.; Hills, D. C.; Hodgin, L. A.; Howard, J. R.; Hume, D. J.; Huntington, J. L.; Husted, R. R.; Isenhart, J. W.; Johnson, R. J.; Kao, W-H.; Killos, P. J.; Lachica, R. D. F.; Linden, J. C.; Nelson, D. K.; Patnode, S. C.; Qureshi, M. C.; Rambo, R. S.; Richardson, L. F.; Rose, R. J.; Sair, R. A.; Scharffenberg, S.; Shonka, M. L.; Smith, R. L.; Stifel, F. B.; Treadwell, G. E. Jr.; Tsuda, Y.; Wise, J. L. Jr.; and Fisher, T. L. (1965) "Identification of a Carboxyl group at the Active Site of Barley MaIt Phosphatase," Proceedings of the Iowa Academy of Science, 72(1), 113-116. Available at: https://scholarworks.uni.edu/pias/vol72/iss1/19 This Research is brought to you for free and open access by the Iowa Academy of Science at UNI ScholarWorks. It has been accepted for inclusion in Proceedings of the Iowa Academy of Science by an authorized editor of UNI ScholarWorks. For more information, please contact scholarworks@uni.edu. Identification of a Carboxyl group at the Active Site of Barley MaIt Phosphatase Authors F. X. Aherne, G. A. Bennett, D. L. Berner, M. E. Burke, N. E. Busch, W. Carrothers, K-P. Chan, A. R. Conradie, J. D. Daniels, F. L. Dunn, M. Feider, L. T. Frobish, M. W. Ganfield, M. E. Hallahan, D. C. Hills, L. A. Hodgin, J. R. Howard, D. J. Hume, J. L. Huntington, R. R. Husted, J. W. Isenhart, R. J. Johnson, W-H. Kao, P. J. Killos, R. D. F. Lachica, J. C. Linden, D. K. Nelson, S. C. Patnode, M. C. Qureshi, R. S. Rambo, L. F. Richardson, R. J. Rose, R. A. Sair, S. Scharffenberg, M. L. Shonka, R. L. Smith, F. B. Stifel, G. E. Treadwell Jr., Y. Tsuda, J. L. Wise Jr., and T. L. Fisher This research is available in Proceedings of the Iowa Academy of Science: https://scholarworks.uni.edu/pias/vol72/iss1/19 1965] FOSSILIFEROUS PLANTS 113 Literature Cited 1. Arnold, Chester A. 1959. Fossil Flora of the Michigan Coal Basin. Univ. of Michigan Press. Ann Arbor, Michigan. pp. 131-269. 2. Bell, W. A. 1938. Nova Scotia Geological Survey Memoir 215. J. 0. Patenaude, I.S.O., Ottawa. 3. -------. 1940. Nova Scotia Geological Survey Memoir 225. J. 0. Patenaude, I.S.O., Ottawa. 4. --------. 1944. Nova Scotia Geological Survey Memoir 238. Edmoncl Cloutier, Ottawa. 5. Canright, James E. 1959. Indiana Department of Conservation and Geo-logical Survey, Report of Progress No. 14. 6. Langford, George. 1958. The Wilmington Coal Flora from a Pennsyl-vania Deposit in Will County, Illinois. Esconi Associates. Identification of a Carboxyl group at the Active Site of Barley Ma It Phosphatase·1 Biochemistry 512 Class of 19652 ( D. J. GRAVES3 , Instructor) Abstract. The dependence of the maximal velocity, Yw with pH for barley malt phosphatase has been studied from pH 4.0 to 5.4. The data show that an ionizable group in the enzyme-substrate complex with a pK .. of 4-4.6 is important for enzymic activity. These results suggest strongly the in-volvement of a carboxyl group of a glutamic or aspartic acid residue at the active site of this enzyme. INTRODUCTION To understand the mechanism of enzyrnic catalysis, it is essential to determine what amino acid residues of the enzyme am involved in the interaction with substrate and to delineate how this interaction results in the formation of free enzyme and product. Although we know rather little about the latter, much information is presently being accumulated about the nature of amino acid residues at the active sites of various enzymes. One approach to this problem is through the study of enzyme kinetics and the pH dependence of the kinetic para-meters, KM and VM. The particular advantages of this probe are that pure enzyme is not essential for this study and the experi-l This researeh was supported by Science Education ( 701-04-30) from the College of Science and Humanities, Iowa State University. •This work was completed during the tenure of the Biochemistry 512 laboratory class, March 9 - May 28, 1965. Coauthors on this paper are: F. X. Aherne, G. A. Bennett, D. L. Berner, M. E. Burke, N. E. Busch, W. Carrothers, V. K-P. Chan, A. R. Gonradie, J. D. Daniels, F. L. Dunn, T. L. Fisher, L. T. Frobish, M. W. Gan-field, M. E. Hallahan, D. C. Hillls, L. A. Hodgin, J. R. Howard, D. J. Hume, J. L. Huntington, R. R. Husted, J. W. Isenhart, R. J, Johnson, W-H. Kao, P. J. Killos, R. D. F. Lachica, J. C Linden, D. K. Nelson, S. C. Patnode, M. C. Qureshi, R. S. Rambo, L. F. Richardson, R. J. Rose, R. A. Sair, S. Scharffenberg, M. L. Shonka, R. L. Smith, F. B. Stifel, G. E. Treadwell, Jr., Y. Tsuda, J. L. Wise, Jr., M. Feider. a Recepient. of a Research Career Development Award of the U.S. Public Health Service ( l-KS-GM-6753-01 ). 1Aherne et al.: Identification of a Carboxyl group at the Active Site of Barley MPublished by UNI ScholarWorks, 1965114 IOWA ACADEMY OF SCIENCE [Vol. 72 mental procedures for such research are rela,tively simple. The major disadvantage is that this procedure results only in pK values and not functional groups. Although the assignment of a functional group from enzyme kinetic data is complicated by the fact that pK values for amino acid residues in proteins may vary with environment ( 1), these assignments. have been in most instances corroborated by other studies ( 2). The present work is with barley malt phosphatase, an enzyme which has a pH optimum of approximately 5.3 ( 3), and in-dicates that loss of enzymic activity on the acid limb of the pH activity curve is due to the protonation of a carboxyl group in the enzyme-substrate complex. MATERIALS AND METHODS Disodium phenylphosphate and Malt diastase (phosphatase) were purchased from Sigma Chemical Co., St. Louis, Missouri. All other chemicals were reagent grade and obtained at the chemistry stockroom at Iowa State University. Enzyme (50 gms) was suspended in 500 mis of distilled water and tpen qeIIJtrifuged to remOVf:l insoluble matter. The super-natant' fluid was dialyzed against three changes of cold distilled water ( 100 ml each) and stored frozen. The adjustment of pH of buffers and substrates were carried out on pH meters with a precision of +0.01 pH unit. For the determination of maximum velocity, VM, at any pH, the following reaction mixtures were prepared: 8 mis of O.OlM ethylenediamine citrate buffer, 1 ml of disodium phenylphosphate (10-1M -- 5 x 10-3M), and 1 ml of enzyme. At various intervals 1 ml aliquots were removed and added to 8 ml of 10% Na2COs to quench the reaction, after which 1 ml of Folin's reagent ( 4) was added for the detection of liberated phenol. After 10 minutes the developed colors were read in a Klett colorimeter utilizing a #66 filter. For each pH, initial velocities were determined for five different substrate concentrations by construction of a tangent at t 0 for each of the progress curves. The maximum velocity was then deduced by extrapolation of a double reciprocal plot of Lineweaver and Burke ( 5) to the velocity at infinite substrate concentration. All kinetic experiments were performed in constant tempera-ture water baths with a temperature control of +0.1°C. RESULTS AND DISCUSSION The dependence of the V M of barley malt phosphatase with pH is illustrated in Figure 1. According to Dixon and Webb (6), ke VM may be expressed by: VM = 1 + 1i + K K ES2 .Es1 H 2Proceedings of the Iowa Academy of Science, Vol. 72 [1965], No. 1, Art. 19https://scholarworks.uni.edu/pias/vol72/iss1/191965] BARLEY MALT PHOSPHATASE 115 -1.B ~ -1.9 ..J -2.0 -2.1 -2.2 4.0 4.2 4.4 4.6 4.B 5.0 5.2 5.4 pH Figure 1. Dependence phosphatase. of the maximal velocity with pH at 37° for barley malt and by analysis of log V M curves against pH, the dissociation constants K Es · and K Es for functional groups in the enzyme-1 2 substrate complex of equation 1 may be determined. The con-stant, k, is a rate constant for the decomposition of the ES com-plex to enzyme and product and e is the total enzyme concentra-tion. The acid limb of the pH activity curve may be expressed by: log VM =log ke +pH - pKEs • Tb,e data show that the slope of the experimental curve ap~ 3Aherne et al.: Identification of a Carboxyl group at the Active Site of Barley MPublished by UNI ScholarWorks, 1965116 IOWA ACADEMY OF SCIENCE [Vol. 72 proaches unit slope at pH's below 4.5 as predicted by the above equation. If the loss of activity on the acid limb was due to an unfolding of the enzyme molecule rather than due to a protona-tion of a specific amino acid residue, it might be expected that the experimental slope would be greater than unity. Davis and Metzler ( 7) observed a slope of approximately three for dependence of KM with pH for threonine dehydrase and postu-lated a reversible denaturation for loss of enzymic activity. For the analysis of pK ES , Dixon and Webb ( 6) showed that l from equation 1 the theoretical curve of unit slope should inter-sect a curve of zero slope and this intersection point should oc-cur 0.3 units above the experimental curve. These data show that a functional group with a pK of 4.5-4.6 is implicated at the active site of barley malt phosphatase (Figure 1). In two other experiments of 37° pK values of 4.4 and 4.6 were obtained. Since the ,B-carboxyl and y-carboxyl group of aspartic and glutamic acid residues, respectively, have pK values from 3-4. 7 ( 6) and no other functional groups in proteins have pK values in this range, it would appear that the observed variation of V M with pH for malt phosphatase is due to the ionization of a carboxyl group. Similar conclusions were reached by Alvarez ( 8) with potato phosphatase. Although the mode of participation of this residue in phosphate ester hydrolyzis is unknown, stucey of models for enzymic reactions shown that carboxy late groups stimulate ester hydrolysis ( 9). In one case a cyclic anhydride has been isolated as an inter-mediate for intramolecular catalysis by a carboxylate group ( 10). Literature Cited 1. Edsall, J. T. and Wyman, J. Biophysical Chemistry, I. Academic Press, New York, New York, 1958). 2. Boyer, P. D., Ann. Rev. Biochem., 29, 15 ( 1960). 3. Olson, W. J., Robbins, G. S., and Dickson, A. D., Am. Soc. Brewing Chemists Proc., 1952 (Barley and Malt Lab., Madison, Wisconsin). 4. Folin, 0. and Ciocalteu, V. J., ]. Biol. Chem., 73, 627 (1927). 5. Lineweaver, H. and Burke, D., ]. Am. Chem. Soc., 56, 658 ( 1934). 6. Dixon, M. and Webb, E. C., Enzymes (Academic Press, New York, New York, 1964). 7. Davis, L. and Metzler, D. E., J. Biol. Chem., 237, 1883 ( 1962). 8. Alvarez, E. F., Biochem. Biophys. Acta, 59, 663 (1962). 9. Bender, M. L., Chem. Rev., 60, 53 ( 1960). 10. Bruice, T. C. and Pandit, V. K., J. Am. Chem. Soc., 80, 5388 ( 1958). 4Proceedings of the Iowa Academy of Science, Vol. 72 [1965], No. 1, Art. 19https://scholarworks.uni.edu/pias/vol72/iss1/19

Identification of a Carboxyl group at the Active Site of Barley MaIt Phosphatase

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Identification of a Carboxyl group at the Active Site of Barley MaIt Phosphatase

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