Enzyme Activities in Bondage? Sirs, In the April issue of BioEssuys, Spivey and Merz(l) provided an excellent discussion of weak enzymeenzyme associations in cells and their putative physiological relevance. Their interpretation of the significance of such associations, which has been similarly emphasized by other workers in this field (cf. ref. 2 for review), is that these associations represent enzyme complexes that enhance metabolic efficiency, as through substrate channeling. However, the authors chose to ‘not discuss’ the potential kinetic and allosteric consequences of enzymatic associations. We feel this is a serious omission, and would like to point out that alternative interpretations of the role of enzyme associations in cells need serious consideration. Rather than enhancing efficiency, weak associations may in some instances function to inhibit enzyme activities in vivo. We have been attracted to this particular view from our studies on enzyme activity in sea urchin eggs and the changes that occur following the stimulation of cell activity at ferti~ization(~). Using unfertilized eggs that have been electrically permeabilized, and which retain much of their cell structure, we find that the activities of a number of enzymes assayed under saturating substrate conditions (i.e. at V,,,) are severely repressed in the permeabilized cells as compared to their activities in homogenates of these same eggs. This inhibition does not result from a limitation on diffusibility of the substrate to the enzyme. However, if the eggs are first fertilized and then permeabilized, we observe a large increase in activity of these enzymes assayed in the permeabilized cells, but not when assayed in the homogenized cell preparations. That is, in spite of a clear increase in cell activity after fertilization, homogenates of both fertilized and unfertilized eggs show the same level of enzyme activities, and these are always much higher than those seen in the permeabilized cells. Assays in permeabilized cells, on the other hand, do show a fertilization-induced, de-inhibition of enzymatic activities, which suggests that some feature of the cell structure must be intimately related to the regulation of the activities of these enzymes in vivo. The regulation of enzyme activity seen in permeabilized cells, but not in homogenates, serves to underline a major point brought up by Spivey and Merz. Enzymeenzyme (or enzyme-structural protein) interactions that are physiologically important in vivo may be intrinsically so weak as to be destroyed by ‘the cataclysmic violence of the most gentle homogenization procedure’ (quote from ref. 4). Intracellular conditions, such as the high local concentrations of proteins and the nonideality of the aqueous solvent, probably enhance the stability of such weak interactions in vivo. In fact, these conditions were shown by Bosca et ul.(’) to influence significantly both the kinetic and the regulatory proper-
ties of rat muscle phosphofructokinase assayed in v i m . If the permeabilized cells retain sufficiently some of these intracellular conditions, then regulatory interactions between enzymes and/or other proteins may be preserved, and therefore become experimentally visible. A conflict between in vitro and in vivo properties of enzyme activities has been seen in other systems. For example, Batke(6) has pointed out that in the glycosomes of Trypanosome species, the predicted maximal enzymatic velocities (extrapolated from (a) the various K,,, values of the glycolytic enzymes determined in their purified forms, and (b) the concentrations of these enzymes) greatly exceed the measured glycolytic fluxes in vivo, and thus the glycolytic pathway in vivo is operating far below the maximal catalytic capacity of the enzymes. Furthermore, Ahn and Klinman(’) have recently reported that the K,,, for dopamine P-monooxygenase from bovine adrenal medulla is 14-40 fold lower when assayed in situ in intact chromaffin granules than when the detergent-solubilized enzyme is assayed in vitro. These analyses, if correct, would indicate that these cells contain an excess of potential enzyme activities, similar to the case with sea urchin eggs, which are largely inhibited or modulated in vivo. Also relevant to this discussion is the provocative hypothesis put forth by Clarke et ul.(*),that suggests a very different role for associations between enzymes and structural proteins in cells. Rather than assuming that the major influence of such binding is on the enzymatic function (i.e., substrate channeling or kinetic modulation), these authors suggest that the reverse process may also be operative in vivo. Specifically,they propose that enzyme binding may influence the organization of structural proteins (e.g., actin, tubulin, etc.) in cells, and may thus integrate the cell’s structural status with its physiological state. Intracellular levels of intermediate metabolites would then be the mediators of this integration. To support their argument, the authors show that actin filaments in vttro are organized to a higher order structure (gelation) by the addition of aldolase, and that this actin-aldolase complex is dissociated by the substrate for aldolase, fructose-l,6bisphosphate. It is possible, therefore, that enzymes may have both catalytic and structural roles in cellular processes. To conclude, while-we agree fully with Spivey and Merz that the evidence supporting the existence of enzyme associations in cells is growing, we do want to urge workers to keep an open mind as to the physiological significance of such interactions. The streamlining of metabolism by substrate channeling is one possibility, and while from an anthropomorphic view it is very appealing, we must remember that enhancing the efficiencies of metabolic pathways may not always be the guiding principle. Regulation of metabolism, where
enzyme associations in vivo serve to restrain enzyme activity, and integration of cell structure with cell function should also be considered when trying to assign a biological ‘reason’ for these enzyme associations. ROBERT R. SWEZEY DAVID EPEL Hopkins Marine Station Stanford University Pacific Grove, CA. 93950
References 1 SPIVEY,H. 0. AND MERZ,J. M. (1989). Metabolic Compartmentation. BioEssays 10, 127-130.
2 SREKE,P. A. (1987). Complexes of sequential metabolic enzymes. Annu. Rev. Biochem. 56,89-124. 3 SWEZEY, R. R. AND EPEL,D. (1988). Enzyme stimulation upon fertilization is revealed in electrically permeabilized sea urchin eggs. Proc. Natl Acad. Sci. USA 85,812-816. 4 MCCONKEY, E. H. (1982). Molecular evolution, intracellular organization, and the quinary structure of proteins. Proc. Natl Acad. Sci. USA 79,3236-3240. 5 BOSCA,L., ARAGON,J. J. AND SOLS, A. (1985). Modulation of muscle phosphofructokinase at physiological concentration of enzyme. J. Biol. Chem. 260,2100-2107. 6 BATKE,J. (1989). Remarks on the supramolecular organization of the glycolytic system in vivo. FEBS Len. 251, 13-16. 7 AHN,N. G. AND KLINMAN, J. P. (1989). Nature of rate-limiting steps in a compartmentalized enzyme system. J. Biol. Chem. 267, 12 259-12 265. 8 CLARKE, F. M., MORTON, D. J., STEPHAN, P. AND WIEDEMANN, J. (1985). The functional duality of glycolytic enzymes: potential integrators of cytoplasmic structure and function. In Cell Motility: Mechanism and Regulation (Ishikawa, H., Hatano, S. and Sato, H., eds.). Tokyo Univ. Press Tokyo.
THE 1991 MIAMI BIO/TECHNOLOGY WINTER SYMPOSIA January 27-February 1,1991 Miami Beach, Florida, USA
“Advances in Gene Technology: The Molecular Biology of Human Genetic Diseases” Organized by
The University Biochemistry & Molecular Biology Foundation, Inc. and Nature Publishing Company Topics Genetic disease genes, Mitochondria1 genes and human genetic disease, DNA sequencing technology, Physical mapping of human DNA, Some specific results from human genetic mapping, Methods for DNA transfer, Cancer related genes, New uses for viral vectors in human genetics, Diagnostic technology for human genetic diseases, Progress toward gene therapy.
If you wish to receive a program, registration forms and poster session information, please write to: The Miami Bio/Technology Winter Symposia, P.O. Box 016129, Miami, FL 33101-6129, USA or call toll free, I-800-MIA-GENE (1-800-642-4363) or fax us at 1-305-324-5665.
ties of rat muscle phosphofructokinase assayed in v i m . If the permeabilized cells retain sufficiently some of these intracellular conditions, then regulatory interactions between enzymes and/or other proteins may be preserved, and therefore become experimentally visible. A conflict between in vitro and in vivo properties of enzyme activities has been seen in other systems. For example, Batke(6) has pointed out that in the glycosomes of Trypanosome species, the predicted maximal enzymatic velocities (extrapolated from (a) the various K,,, values of the glycolytic enzymes determined in their purified forms, and (b) the concentrations of these enzymes) greatly exceed the measured glycolytic fluxes in vivo, and thus the glycolytic pathway in vivo is operating far below the maximal catalytic capacity of the enzymes. Furthermore, Ahn and Klinman(’) have recently reported that the K,,, for dopamine P-monooxygenase from bovine adrenal medulla is 14-40 fold lower when assayed in situ in intact chromaffin granules than when the detergent-solubilized enzyme is assayed in vitro. These analyses, if correct, would indicate that these cells contain an excess of potential enzyme activities, similar to the case with sea urchin eggs, which are largely inhibited or modulated in vivo. Also relevant to this discussion is the provocative hypothesis put forth by Clarke et ul.(*),that suggests a very different role for associations between enzymes and structural proteins in cells. Rather than assuming that the major influence of such binding is on the enzymatic function (i.e., substrate channeling or kinetic modulation), these authors suggest that the reverse process may also be operative in vivo. Specifically,they propose that enzyme binding may influence the organization of structural proteins (e.g., actin, tubulin, etc.) in cells, and may thus integrate the cell’s structural status with its physiological state. Intracellular levels of intermediate metabolites would then be the mediators of this integration. To support their argument, the authors show that actin filaments in vttro are organized to a higher order structure (gelation) by the addition of aldolase, and that this actin-aldolase complex is dissociated by the substrate for aldolase, fructose-l,6bisphosphate. It is possible, therefore, that enzymes may have both catalytic and structural roles in cellular processes. To conclude, while-we agree fully with Spivey and Merz that the evidence supporting the existence of enzyme associations in cells is growing, we do want to urge workers to keep an open mind as to the physiological significance of such interactions. The streamlining of metabolism by substrate channeling is one possibility, and while from an anthropomorphic view it is very appealing, we must remember that enhancing the efficiencies of metabolic pathways may not always be the guiding principle. Regulation of metabolism, where
enzyme associations in vivo serve to restrain enzyme activity, and integration of cell structure with cell function should also be considered when trying to assign a biological ‘reason’ for these enzyme associations. ROBERT R. SWEZEY DAVID EPEL Hopkins Marine Station Stanford University Pacific Grove, CA. 93950
References 1 SPIVEY,H. 0. AND MERZ,J. M. (1989). Metabolic Compartmentation. BioEssays 10, 127-130.
2 SREKE,P. A. (1987). Complexes of sequential metabolic enzymes. Annu. Rev. Biochem. 56,89-124. 3 SWEZEY, R. R. AND EPEL,D. (1988). Enzyme stimulation upon fertilization is revealed in electrically permeabilized sea urchin eggs. Proc. Natl Acad. Sci. USA 85,812-816. 4 MCCONKEY, E. H. (1982). Molecular evolution, intracellular organization, and the quinary structure of proteins. Proc. Natl Acad. Sci. USA 79,3236-3240. 5 BOSCA,L., ARAGON,J. J. AND SOLS, A. (1985). Modulation of muscle phosphofructokinase at physiological concentration of enzyme. J. Biol. Chem. 260,2100-2107. 6 BATKE,J. (1989). Remarks on the supramolecular organization of the glycolytic system in vivo. FEBS Len. 251, 13-16. 7 AHN,N. G. AND KLINMAN, J. P. (1989). Nature of rate-limiting steps in a compartmentalized enzyme system. J. Biol. Chem. 267, 12 259-12 265. 8 CLARKE, F. M., MORTON, D. J., STEPHAN, P. AND WIEDEMANN, J. (1985). The functional duality of glycolytic enzymes: potential integrators of cytoplasmic structure and function. In Cell Motility: Mechanism and Regulation (Ishikawa, H., Hatano, S. and Sato, H., eds.). Tokyo Univ. Press Tokyo.
THE 1991 MIAMI BIO/TECHNOLOGY WINTER SYMPOSIA January 27-February 1,1991 Miami Beach, Florida, USA
“Advances in Gene Technology: The Molecular Biology of Human Genetic Diseases” Organized by
The University Biochemistry & Molecular Biology Foundation, Inc. and Nature Publishing Company Topics Genetic disease genes, Mitochondria1 genes and human genetic disease, DNA sequencing technology, Physical mapping of human DNA, Some specific results from human genetic mapping, Methods for DNA transfer, Cancer related genes, New uses for viral vectors in human genetics, Diagnostic technology for human genetic diseases, Progress toward gene therapy.
If you wish to receive a program, registration forms and poster session information, please write to: The Miami Bio/Technology Winter Symposia, P.O. Box 016129, Miami, FL 33101-6129, USA or call toll free, I-800-MIA-GENE (1-800-642-4363) or fax us at 1-305-324-5665.
