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A M . ZOOLOGIST, 11:425-485 (1971). Enzyme Mechanisms in Temperature and Pressure Adaptation of Off-Shore Benthic Organisms: The Basic Problem PETER W. HOCHACHKA Department of Zoology, University of British Columbia, Vancouver 8, B. C, Canada SYNOPSIS. The degree of participation of any given enzyme reaction in metabolism is not determined by energy-volume relationships, but rather by enzyme affinities for key regulatory ligands. In temperature adaptation, enzyme-substrate affinities often decrease as thermal kinetic energy increases, thus compensating for temperature-induced changes in reaction velocity at low substrate concentrations. Since the effects of pressure are not unidirectional (some enzymes are accelerated; some are unaffected; some are decelerated), a more functional solution in pressure adaptation is to elaborate enzymes whose affinities for key metabolites are pressure independent. In consequence, at physiological substrate concentrations, the pressure sensitivities of catalysis and control of catalysis are probably held at a minimum. restricted and often must occur at the molecular, rather than the whole-animal, When an organism encounters an al- level of organization. A moment's contemtered environment, its metabolic response plation will make this distinction clear. An to that change is "adaptive" if in proper artic marine mammal, for example, cosequence it activates an appropriate set of ordinates a whole series of physiological reactions for answering the cell's new car- processes in order to maintain a constant bon and/or energy demands. For example: body temperature in the face of the ex(1) the glucocorticoid-controlled produc- treme outer one, while, in contrast, an artion of glucose during periods of fasting, tic fish, because it cannot regulate its body but not during satiated periods; (2) the temperature, must accept the environmenactivation of lipolysis in adipose tissues tal one as given. Here, temperature is imduring thermogenesis, but not during peri- pinging directly upon enzyme reaction ods of thermal balance; (3) the activation rates and the organism's adaptive responses of the urea cycle enzymes in Amphibian must begin at this point. The same is true liver during terrestrial phases of life but for the effects of pressure on organisms in not during aquatic larval stages. All such the water column. categories of metabolic response can be Biologists have known for many years said to be patently adaptive. Formally, the that the metabolism of many poikilosignal and the precise response are the two therms is carefully adjusted to the thermal components of the system that differ from regime in which they live. To choose an example to example, but in each example extreme example, rates of energy metabowe are basically dealing with a network of lism, swimming performance, and growth reactions whose degree of participation in of antartic fishes are as high at near 0°C as metabolism at any time is automatically those of temperate zone fishes at 15-25°C and usually autocatalytically controlled. By (Wohlschlag, 1961; Somero et al., 1968). means of such control systems, most orga- This extreme cold adaptation of metabolic nisms gain a certain degree of indepen- processes presumably requires evolutiondence from the outer environment. In ary time and has never been observed durwarm blooded forms this is manifest as ing shorter term adjustments, such as occur homeothermy; in cold blooded forms, the during seasonal acclimatization (or acclidegree of independence from such parame- mation) of eurythermal temperate zone ters as temperature or pressure is more fishes. Although the magnitude of the acINTRODUCTION 425 426 PETER W. HOCHACHKA en O FIG. 1. Rates o£ O2 uptake by minced tuna red muscle (T-RM), tuna white muscle (T-WM) and skeletal muscle from the white rat (Rat-M). Data redrawn from Cordon (19G8). Rates of CO, production from aceta te-1-C11 oxidation by trout liver slices at different temperatures are shown in the middle curve (x symbols). O2 uptake is in mm'/gm/hour. C"O2 production is in picocuries/mg protein/hour. Data redrawn from Dean. (1969). climation response is less marked, it is in the same direction; thus, the respiration of tissues from cold acclimated fish is usually higher than that of warm acclimated ones at all temperatures examined (see Precht, 1968; Fry and Hochachka, 1970). Finally, a number of examples of temperature independent metabolic processes have been discovered in poikilothermic organisms, and these represent another category of thermal compensation. Glycogen synthesis in insects can be temperature-independent over quite broad thermal ranges (van Handel, 1966); O2 uptake by intertidal organisms can display Q l n values of near unity (Newell, 1966); the respiration rate of tuna muscle minces (Gordon, 1968) and the oxidation of acetate-1-C14 by salmonid tissues (Dean, 1969) are stable over 20-30°C ranges (Fig. 1). Such immediate thermal compensation is surely as dramatic as the extreme cold adaptation of metabolism seen in the antarctic fishes. For convenience we can distinguish three time-courses of temperature adaptation in poikilotherms: (1) evolutionary adaptation, (2) thermal acclimation, and (3) immediate thermal compensation. Comparable time-courses of exposure to high and/or variable pressures are probably encountered by organisms in the marine water column. However, studies on the effects of long-term exposures to high piessutt's are not abundant, mainly be- 427 THE BASIC PROBLEM u o 2 - 330 353 5 10 / FIG. 2. Arrhenius plot of liver citrate synthase activities from cold (2°C) acclimated trout at saturating oxaloacetate and acetylCoA concentrations is shown in the curve labeled Vmax. The calculated activation energy is 8.8 kcal/mole, corresponding to a Qio of about 1.7. In the two lower curves, oxaloacetate concentrations were at saturating levels, but acetylCoA levels were 0.01 and 0.023 raM as shown. It is evident that the Qlo is dramatically decreased at low substrate concentrations. Data redrawn from Hochachka and Lewis (1970). Similar data are available for several different enzyme systems and are discussed by Hochachka and Somero (1971). cause the requisite technology is unavailable. For this reason, I will restrict this paper to immediate effects of temperature in comparison with the effects of pressure upon enzymes of deep-sea organisms. given in °K. A typical graphical presentation of this equation is shown in Figure 2 (for curve labeled, Vmad;). For workers interested in temperature adaptation, two portions of the Arrhenius curve are of interest—the activation energy which is directly proportional to the slope of the line, and the thermal optimum. Earlier workers considered that either characteristic or both should correlate with the habitat temperature, but as more data became available, it became evident that neither does (see Hochachka and Somero, 1971). In regard to thermal optima, the argument is simply that for most enzymes these are far beyond the lethal limits for the organism. As regards the value of /x, the situation is somewhat less clear. Basically, the idea commonly held is that selection would favor en- IMMEDIATE EFFECTS OF TEMPERATURE ON POIKILOTHERMIC ENZYMES Classically, the effect of temperature on enzyme reaction rates was interpreted according to the Arrhenius relationship given by equation (1): * = 2.3R log KTi — log KT(> 1 i — 1 (1) o where ^ is the activation energy, R the gas constant, K the rate constant for the reaction at 2 different temperatures, T1 and To, 428 PETER W. HOCHACHKA CITRATE SYNTHASE 1 - FIG. 3. Effect of temperature on the Km of acetylCoA for liver citrate synthase from cold acclimated rainbow trout. Data redrawn from Hochachka and Lewis (1970). As temperature in- creases, the apparent enzyme-substrate affinity (reciprocal of the Km) decreases, thus compensating the reaction for changes in the thermal energy of the reactants. zymes of high catalytic efficiency (and hence low ji value) in organisms living at low temperatures. Where JX is an acceptable measure of catalytic efficiency, the correlation may indeed hold. But the turnover number of an enzyme can also be strongly influenced by activation entropy, which has been largely ignored by earlier studies. This may account for the absence of an obvious correlation between the ^ value for any two homologous enzymes and the thermal habitat of the parent species, whereas such a correlation does appear to exist for the turnover number of enzymes. (See Hochachka and Somero, 1971, for a further discussion of this point.) The situation, however, is still more complex. As it> e\ident in Figure 2, the slope of Arrhenius plots can vary dramatically depending upon substrate concentra- tions. At low substrate levels, the temperature coefficient (Qlo) is dramatically reduced and can take on values of less than 1.0. The explanation for such seemingly anomalous thermal properties is to be found in the nature of the catalyst. For a large number of enzyme systems, we have found that apparent Michaelis constant (Km) varies directly with temperature over at least a part of the biological range. Over this range, as temperature increases, the apparent enzyme-substrate affinity decreases in such a way as to compensate for the thermal energy change (Fig. 3). Thus, under low (physiological) substrate concentrations, the reaction rate is being determined by the kinetic properties of the catalyst rather than thermudyniunit parameters (Hochachka and Somero, 1971). In many wa)s, there is a strong similarity T H E BASIC PROBLEM 429 FIG. 4. Comparison of the effects of the positive modulator, AMP, and of low temperatures upon the activity of king crab phosphofructokinase. Data redrawn from Freed (1971). between the effects of temperature and the effects of metabolite modulators on enzyme-substrate affinities. A particularly clear example is to be found in the case of king crab phosphofructokinase, where the effects of low temperature and of the positive modulator, AMP, are strikingly comparable (Freed, 1971) (Fig. 4). In this example, as in the case of many regulatory enzymes, catalytic activity is regulated by the binding of specific metabolites at sites separate from the catalytic site; usually, these modulators alter the apparent enzyme-substrate affinity with no necessary effect on the maximal velocity (Vmaa:) of the reaction. Positive modulators decrease the apparent Km, and sometimes convert the sigmoidal curve into a hyperbolic one. (At low substrate levels, this is tantamount to activating the enzyme.) In both these effects, the action of positive modulators is analogous to that of low temperature. Somero (1969) also has described an elegant example of this in the case of king crab pyruvate kinase (PK): At low temperatures, the apparent Km of the substrate (PEP) is reduced, and substrate saturation curves become hyperbolic rather than sigmoidal. Another fundamental characteristic of regulatory enzymes is that saturation curves for positive or negative modulators also are often sigmoidal. An important implication of this is that small changes in modulator concentration can lead to relatively large changes in the activity of the enzyme. This property coupled with others, such as product activation, leads to regulatory behavior which approximates an "on-off" switch. In general, "on-off" switch mechanisms of poikilothermic enzymes seem to be less temperature sensitive than are enzyme- 430 PETER W. HOCHACHKA substrate interactions (see Hocliachka and Somero, 1971). This admirably suits poikilothermic existence, for the regulation of enzyme activity can then be achieved with equal efficiency over the entire biological temperature range, irrespective of the effects of temperature on maximum velocities. Because of the fundamental role of Km modulation in enzyme regulation in general, then, it is not too surprising that the effects of change in the thermal energy of reactants can be counteracted by appropriate and instantaneous effects of temperature, presumably upon enzyme conformation and hence upon enzyme-substrate affinities. But, such actions of temperature are complicated in deep-sea fishes by continuous high pressures and in midwater fishes by linear changes in pressure encountered during vertical migrations through the water column. Hence, it is informative to examine the effects of pressure on enzymes in the context discussed above. IMMEDIATE EFFECTS OF PRESSURE ON ENZYME REACTION RATES The basic action of pressure upon various metabolic processes is fairly well documented. Nearly 40 years ago, Johnson and Eyring and coworkers described the fundamental theorem relating pressure and reaction velocities (see Johnson and Eyring, 1970). This relationship is given in equation (2): AV*=2.3RT (2) where AJ7* is the volume change of activation, R the gas constant, K the velocity constant at pressure px and p2. This equation for the relation between pressure and reaction rate is of the same form as the Arrhenius equation for the relation between temperatures and rate. According to the Arrhenius theory of absolute rates, it is necessary to assume the existence of an activated complex to account for the large effect of temperature on reaction rates. In- deed, the reaction rate can be taken as a measure of the concentration of the activated complex. So long as the activated complex represents a more energy-rich configuration than the average value of its constituents, a rise in temperature will increase the rate. Where this is no longer true, the rate process goes through a maximum. A comparable view holds with regard to pressure. Here, A P can be taken as the ratio volume of the activated complex volume of the reactants So long as the volume of the activated complex exceeds the average volume of its constituents outside the complex, pressure retards the reaction. At the point where the volumes become equal, there is no change in the reaction rate under pressure. When the volume of the activated complex is less than that of the reactants, pressure accelerates the reaction rate (Fig. 5). In this latter case, the reaction rate can go through a pressure optimum, the actual pressure at which the optimum occurs often depending upon the temperature (Fig. 5). Most previous workers in this field have obtained results comparable to those shown in Figure 5 (see Morita, 1967; ZoBell, 1970; Johnson and Eyring, 1970) and this seems true even at higher levels of organization. (See Brown et ah, 1958, for effects of pressure on tension developed in turtle heart at different temperatures.) The above noted interaction between temperature and pressure is an important aspect of this problem but one which often has been given inappropriate consideration. ZoBell and Hittle (1969) for example found that the rate of starch hydrolysis by more than a hundred species of marine bacteria is roughly proportional to their rate of growth at different pressures and temperatures. To gain further information on this matter, these workers then examined a partially purified a-amylase prepared from Bacillus sitbtilis, a well studied non-marine bacterium! This enzyme was found to be accelerated by "deep-sea pressures" at temperatures be- T H E BASIC PROBLEM 431 PRESSURE FIG. 5. Steady state levels o£ luminescence intensity in cell free extracts of the luminous bacterium Achromobacter fishcheri. Data redrawn from Strehler and Johnson (1954). See also Johnson and Eyring (1970) for recent discussion of this system. tween 25-60°C, but not at lower temperatures {see also ZoBell, 1970). The authors point out that such acceleration is predicted by theory since starch hydrolysis is a reaction that takes place with a volume decrease. But starch hydrolysis in marine bottom bacteria occurs at 2-4°, where no pressure activation was noted; hence, either the data or the theory are inadequate. The authors further point out that, whereas "deep-sea pressures" inactivate the enzyme at temperatures below 35°C, they stabilize it at temperatures between 4560°C. The implication is that such pressure-stabilization is of some biological significance. Since nowhere on this planet do temperatures of the ocean bottom ever come close to 45°C, their results indicate (1) that the enzyme characteristics measured are biologically meaningless, or (2) that the homologous enzyme from ocean bottom bacteria must display entirely different properties—or both. This field is riddled with examples of a similar kind (see review by Morita, 1967). Even Schleiper (1968), who is quite aware of this problem, concluded that because the pressure resistance of tissue of Mytilus eclulis is greater at 20° than at 10°, deep-sea animals would cope more successfully with ocean bottom pressures at higher temperatures. Possibly this is true, but certainly it is biologically irrelevant. The point is that temperature of the ocean bottom is 2-4°C; this is where off-shore benthic organisms live, and within the group this is where adaptation to pressure, if it is going to occur, must occur. Much of the available literature also stresses the above formal analogies between the equations relating pressure and temperature to reaction rates of various metabolic processes (equations 1 & 2 above). However, I think that it is important to stress the following difference. Whereas temperature affects all chemical reactions in the same way (by altering the kinetic energy of the reactants), pres- 432 PETER W. HOCHACHKA G1P Si- 1 - 7 -3.4 Hk - 5.0 G6P '•••"•"••|ii'"""iiti1111"11"1*1* 6PG GLUCOSE G6PDH G6 Pase - 4.0 2: §5 si i F6P FIG. 6. The G6P crossroads in metabolism. Each of the enzymes involved directly in G6P production or utilization is shown in the Figure along with the standard free energy change (AG°) which accompanies each reaction. Phosphoglucomutase (PGM); hexokinase (Hk); glucose-6-phosphatase (G6Pase); hexose isomerase (isomerase); and glucoses-phosphate dehydrogenase (G6PDH) are the abbreviations used. Three of these enzymes (Hk, G6Pase, and G6PDH) are known to be involved in the partitioning of G6P between these various reaction pathways. sure activates some, retards some, and does not affect others. What is more, pressure can bring about all three of these effects on a given enzyme-catalyzed reaction depending upon the temperature, or more precisely, upon the enzyme conformation adopted at different temperatures. It is evident, therefore, that from a functional and evolutionary point of view, pressure is an entirely different kind of physical parameter than is temperature. This conclusion has some interesting implications. Consider for example the effects of pressure on a branch point in metabolism such as the G6P crossroads (Fig. 6). In theory, AV* for each reaction could be calculated from knowledge of the geometry of the initial and transition state of the reacting components. In practice, of course, sufficient information about the configuration of the transition state and its neighboring molecules is a\ailable for only the simplest of reactions, and is certainlv unavailable for the reactions of G6P metabolism. Hence, to predict the sign and magnitude of AV* for such reactions approximations have to be used. These usually assume AV* to be the sum of two terms: the "structural" term, A\V*, is the change in volume of die reacting molecules during bondbreakage and bond-formation; the "solvation" term, A^V*, is the accompanying volume change due to interaction with surrounding molecules. When ionic species are formed or disappear in the transition state, the "solvation" contribution to AV* usually excedes the "structural" volume change in the reacting molecules. As a first approximation, then, we may conclude that, in the absence of regulation on the part of the organism, the "charge type" of the reaction will determine the effect of pressure upon its rate. For a branchpoint such as the G6P crossroads (Fig. 6), the ionic species invohed in THE BASIC PROBLEM each reaction as well as the reaction mechanisms are specific to each competing pathway. In addition to different "solvation" contributions to AJ7* for each of the G6P reactions, each is catalyzed by different kinds of enzymes (some are membrane bound; some are apparently free in solution; some are single-subunit types; some are oligomeric). For these reasons, the effects of pressure upon each of these reactions will be quantitatively different. Now three of the enzymes (Hexokinase, G6Pase, G6P dehydrogenase) undoubtedly are involved in regulation of G6P metabolism in liver (Scrutton and Utter, 1968; Atkinson, 1966). Hence, even in such a simple case, it is perfectly clear that the flow of carbon through metabolic branchpoints may be profoundly affected by pressure changes and therefore that the control requirements at such points may depend critically upon the pressure. For the same reasons, the effects of pressure upon the partitioning of carbon and energy between various metabolic pathways may be extreme. Some suggestive data are available in the literature. Chumak (1959) observed that in a marine bacterium both the uptake and the manner in which glucose is metabolized vary dramatically with pressure: CO2 production from glucose is inhibited by pressure, whereas glucose uptake and the production of acid end products both increase markedly. Similarly, the extensive studies of pressure effects on developing marine eggs demonstrate that the partitioning of carbon and energy between DNA synthesis, formation of the mitotic spindle, chromosomal movements, and cytokinesis is markedly affected by pressure (see Zimmerman, 1970) . But, these studies and a host of others of their type (see ZoBell, 1970; Morita, 1967) were designed more from an empirical than from a functional point of view; hence, interpretation of the data in mechanistic terms is impossible. In any event, it is clear that on theoretical grounds, basic problems of integrating various effects of pressure suggest essential requirements for pressure adaptation at the molecular level. 433 The wide abundance of benthic and mesopelagic organisms which thrive under high and/or variable pressures indicate that the enzymatic problems imposed by this parameter are circumvented in nature. The question is how. Most earlier studies of pressure effects on enzymes (often using enzymes from mammalian and nonmarine bacterial sources) have measured pressure effects on catalysis under conditions of saturating substrates, cofactors, and/or coenzymes. And, indeed, the formal analogies between the effects of temperature and pressure are valid only under these conditions, because both the Arrhenius and the Johnson-Eyring equations (equations 1 and 2 above) assume a saturated catalyst. Unfortunately, it is now almost axiomatic in the literature on metabolic control that in vivo most enzymes never "see" saturating concentrations of substrates (Atkinson, 1969; Scrutton and Utter, 1968). As already mentioned, under in vivo conditions, regulation of enzyme activity is usually achieved through the control of enzyme affinities for unique ligands (substrates, cofactors, modulators). Hence it is important to have information on the effects of pressure on these parameters. In this context, our initial studies of fructose diphosphatase (FDPase) in the liver of deep-sea fish indicate that affinities of the enzyme for its substrate (FDP), its cofactor (Mg2+) and its negative modulator (AMP) are all independent of pressure (Hochachka et al, 1970). This result is particularly relevant from a functional point of view, for it suggests that at low substrate concentrations control of catalysis is pressure independent, irrespective of what pressure does to the maximum velocity. In the studies presented in this report, this general conclusion is fully confirmed for several enzyme systems. When the opportunity arose to extend our studies on enzymes from deep-sea fish, we chose a series of enzymes whose physiological functions and regulatory properties arc fairly well documented. These enzymes, the reactions they catalyze, and their 434 PETER W. HOCHACHKA G6P F6P High ATP Low AMP 1 TP NAD—i NADH # PEP NADH NAD PYR LAC FIG. 7. A number of key steps and control circuits in glycolysis. Phosphofructokinase (PFK), which catalyzes a transphosphorylation of F6P thus forming FDP, is inhibited by energy-saturating conditions (high ATP) while the reverse reaction, catalyzed by fructose diphosphatase (FDPase), is simultaneously deinhibited. Under energy-depleted conditions, high le\els of AMP inhibit FDPase and concurrently activate PFK. The pyrmatc kinase (PK) catalyzed conversion of PEP to pyruvate (pyr) is likewise under stringent regulation by feedforward FDP activation and feedback ATP inhibition. The comersion of pjr to lac (lactate), catalyzed by lactate dchydrogenase isozymcs, can be involved in the regulation of gHcolysis by affecting the supply of NAD. The effects of pressure on FDPase, PKK, PK, and I.DHs will be examined in the companion papers. integration into glycolysis and energy metabolism as a whole are diagrammed simply in Figure 7. More detailed properties are described in the companion papers. At this point, what I want to stress is that in benthic fishes, whether pressure accelerates THE BASIC PROBLEM maximum velocities (as in the case of phosphofructokinase and FDPase), does not change them (as in the case of LDH) or retards them (as in the case of PK), enzyme-substrate and enzyme-modulator affinities are largely insensitive to pressure. These studies o£ temperature and pressure effects on poikilothermic enzymes lead to a unifying hypothesis; namely, that at physiological substrate concentrations enzyme reaction rates are controlled by key kinetic parameters rather than by energyvolume relationships. In the case of temperature adaptation, enzyme-substrate affinities often are adjusted to compensate for the unidirectional effect of thermal change. In pressure adaptation, because the effects of pressure are not unidirectional, a more functional solution to the problem is to maintain enzymes whose affinities for their substrates are pressure-independent. In consequence, at physiological substrate concentrations, the pressure sensitivities of catalysis and control of catalysis are probably held at a minimum. If, in regard to pressure, these catalytic and regulatory properties are widespread in enzymes ot benthic and mesopelagic organisms, they supply a sufficient mechanism for avoiding uncontrollable effects of high pressure on chemical reactions. REFERENCES Atkinson, D. E. 1966. Regulation of enzyme activity. Aiinu. Rev. Biochem. 35:85-124. Atkinson, D. E. 1969. Limitation of metabolite concentrations and the conservation of solvent capacity in the living cell. Curr. Top. in Cell. Regul. 1:29-43. Brown, D. E. S., K. F. Cuthe, H. C. Lawler, and M. P. Carpenter. 1958. The pressure, temperature and ion relations of myosin ATPase. J. Cell. Comp. Physiol. 5259-77. Chumak, M. D. 1959. The effect of high pressure on the rate of glucose utili/.ation by barotolerant bacteria. Dokl. Rlol. Sci. 126:524-526. Dean, ]. M. 1969. The metabolism of tissues of thermally acclimated trout (Salmo gairdneri). Comp. Biochem. Physiol. 29:185-196. Freed, J. M. 1971. 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