Proc. Nat. Acad. Sci. USA Vol. 72, No. 8, pp. 3014-3018, August 1975 Biochemistry Activation volumes in enzymic catalysis: Their sources and modification by low-molecular-weight solutes (water-protein interactions/protein conformational changes/hydrostatic pressure effects on enzymes) PHILIP S. LOW AND GEORGE N. SOMERO Scripps Institution of Oceanography, University of California, San Diego, Box 1529, La Jolla, Calif. 92037 Communicated by A. A. Benson, May 22, 1975

ABSTRACT Changes in enzyme conformation are often the presence of different low-molecular-weight solutes, we accompanied by large changes in volume. Model compound have demonstrated that the conclusions based on model studies suggest that these volume changes may derive from two sources: (1) "hydration density" effects due to changes in transfer studies are valid in the case of catalytic conforma- the exposure to solvent of protein groups which modify water tional changes. density, and (ii) "structural" contributions arising from In the second paper in this series (10), we will discuss the changes in the volume of the protein itself. energetic costs of conducting these conformational (volume) An experimental approach was developed to test the valid- changes and present a new mechanism for explaining (I) cat- ity of the predictions based on model compound studies for alytic rate-enhancement by enzymes, and (if) ion activation catalytic conformational changes. By examining the effects of different solutes on the activation volumes of different en- and inhibition of enzymic reactions. zymic reactions, we show that both sources of volume change provide significant contributions to the activation volume. Changes in enzyme conformation, such as those which ac- EXPERIMENTAL SECTION company catalysis, often occur with substantial volume 1. Theory. The pressure dependence of catalytic rates has changes (1-3). An understanding of the factors responsible been described in detail by Laidler (11). Under conditions of for these volume changes therefore may be of considerable saturating substrate concentrations, the pressure dependence importance in advancing our knowledge of the structural of reaction velocity is due entirely to the activation volume bases and energetic consequences of conformational of the reaction, assuming that the enzyme is not subject to changes. denaturation by the increased pressures. The basic equation Brandts, Kauzmann, Scheraga, and Hvidt and their co- relating reaction velocity to pressure is, workers (4-9) have discussed protein volume changes, espe- cially those which occur during denaturation, in terms of k -koe.PAV+/RT model compound studies. In these model studies, protein group (aminoacid side-chain and peptide linkage) analogues where kp is the velocity at a gauge pressure P (in atmo- are transferred from nonpolar to aqueous solvents in an at- spheres), ko is the corresponding velocity at 1 atm pressure, tempt to simulate the transfer of a protein group from the and R is the gas constant, equal to 82 cm3 atm/0K mol. protein interior to water during a conformational change. Activation volumes can be computed from the slope of a Large volume decreases frequently accompany transfer of plot of the logarithm of the maximal velocity (V..) of the. protein group analogues to water, and these are thought to reaction as a function of pressure (2). This procedure is for- be due to constriction of water around the protein group in mally analogous to the Arrhenius plot technique for deter- the aqueous phase (4-9). These authors have also suggested mining activation energies, as the equation below indicates: that a second source of volume change during denaturation might derive from changes in the "packing efficiency" of AV+ = L 2-lnk1 amino acids accompanying the change in protein structure. _R The studies described in this paper provide an empirical test of the validity of these conclusions in terms of their ap- where k2 and k1 are the reaction velocities at pressures P2 plication to catalytic volume changes. Specifically, we exam- and PI, respectively. ined the volume change which occurs when the enzyme- 2. Determination of A V4. Substrate saturation curves substrate ground-state complex is activated to the transition were generated for each enzyme at 1, 204, 408, and 612 atm state. The volume change which occurs during this activa- pressure, using at least seven concentrations of substrate at tion event is termed the "activation volume" (AV*) and each pressure. Maximal velocities were determined by com- equals the volume of the system containing the activated puter from these data according to the method of Woolf complex (VEST) minus the volume of the system containing (12). The activation volume for each reaction was then com- the ground-state complex (VES). By modifying these activa- puted from the slope of the plot of log Vm,,. against pressure, tion volumes through conducting the enzymic reactions in as discussed above. In all assays where salt concentration was varied, the sub- strate concentration representing Vm. was chosen according Abbreviations: PK, pyruvate kinase; LDH, lactate dehydrogenase; to the following two criteria. First, the Vow concentration MDH, malate dehydrogenase; IDH, isocitrate dehydrogenase. had to lie on the plateau region of the saturation curve for 3014 Downloaded by guest on September 30, 2021 Biochemistry: Low and Somero Proc. Nat. Acad. Sci. USA 72 (1975) 3015 all pressures such that small variations in substrate concen- AV*= Vj- VE tration would not affect the rate of the reaction. Second, log Vmax had to vary linearly with pressure over the entire A. "Hydration density" contribution to LV; range of pressures used to ensure that no pressure-induced denaturation occurred. All components of the assay system except enzyme and salt were mixed together in a stock assay solution to elimi- nate effects of pipetting errors. All activity assays were per- formed in a Pye-Unicam SP-1800 UV-visible spectropho- tometer equipped with an Aminco pressure cell (13). The desired pressure was reached within 15 sec in all cases after the addition of enzyme to start the reaction. The tempera- ture of the assay solution was controlled to within 40.20C. Pyruvate kinase (PK) was assayed at 50C, while lactate de- B. "Structural contribution to AV* hydrogenase (LDH), malate dehydrogenase (MDH), and isocitrate dehydrogenase (IDH) were assayed at 15'C. These ES - temperatures were chosen in part to avoid the denaturation >E that occurred at higher temperatures. 3. Enzymes and Assay Solutions. (a) Pyruvate kinase (EC 2.7.1.40; ATP:pyruvate 2-O-phosphotransferase). PK was purified to homogeneity from white skeletal muscle of the cold-water marine teleost Scorpaena gutatta according to a modified version of the procedure of Bondar (14). The enzyme was highly stable under pressure and displayed FIG. 1. A diagrammatic illustration of the two proposed pressure responses qualitatively similar to those observed for sources of activation volume. other pyruvate kinases examined (1). (d) Lactate dehydrogenase (EC 1.1.1.27; lactate:NAD+ The PK assay mixture contained, in a total volume of 5.0 oxidoreductase). Purified rabbit M4 LDH was purchased ml, 0.60 mM phosphoenolpyruvate (tricyclohexylammon- from Calbiochem. The assay solution contained, in a total ium salt, Sigma Chemical Co., St. Louis, Mo.), 10 mM volume of 5.0 ml, 2 mM pyruvate, 0.15 mM NADH, and 25 MgCl2, 1 mM ADP (disodium salt, Sigma), 0.15 mM NADH, mM Tris.HCl buffer, pH 8.0 at the assay temperature. The excess LDH activity (Calbiochem, La Jolla, Calif.; rabbit M4 reaction was initiated by addition of enzyme and followed as isozyme), 50 mM Tris-HCl buffer, pH 7.5 at the assay tem- described above for PK. perature; and varying concentrations of salts as indicated in the figures and tables. The activation volume of the PK RESULTS AND DISCUSSION reaction is independent of pH over the pH range 7-9 (13). The two proposed sources of activation volume are illus- The reaction was initiated by addition of PK and was fol- trated diagrammatically in Fig. 1. The "hydration density" lowed spectrophotometrically by recording the decrease in source results from the movement of water-density-modi- absorbance at 340 nm as a function of time. fying protein groups into or away from contact with water. PK from other sources was assayed as above. The enzyme The model transfer processes listed in Table 1 are suggested preparations used were either 1:5 (weight:volume) muscle to simulate such movements as they might occur at the en- homogenates that had been centrifuged at 12,000 X g for 20 zyme-water interface during catalysis. Since complete expo- min in the cases of Katsuwonus pelami (skipjack tuna), sure or withdrawal of protein groups during catalysis is un- Cancer magister (marine crustacean), and Trematomus likely, the volume changes for the model transfer processes (Pagothenia) borchgrevinki (Antarctic teleost) or were puri- (AVt) (Table 1) represent an upper limit to each type of fied enzymes in the cases of rabbit, Mugil cephalus (mullet transfer which might occur during a catalytic conformation- fish), Bufo nwrinus (toad), and chicken. al change. Thus, for example, the exposing of a methyl side- (b) Malate dehydrogenase (EC 1.1.1.37; L-malate:NAD+ chain of an alanyl residue during the formation of the transi- oxidoreductase). Purified pig heart MDH (Calbiochem) was tion state might reduce AV* by as much as 22 cm3/mol. assayed as follows. The assay system contained, in a total Because certain salts are known to modify the volume volume of 5.0 ml, 0.15 mM NADH, 0.2 mM oxaloacetate changes accompanying the model transfer processes* (20- (prepared fresh each day), 40 mM Tris-HCl buffer, pH 7.8 22), we predicted that transfer processes accompanying an at the assay temperature, and varying concentrations of enzymic conformational change also should be sensitive to salts. The reaction was initiated by adding enzyme and fol- salts. The absence of salt effects on AV* would indicate that lowed as described above. changes in the exposure of aminoacid side-chains and pep- (c) Isocitrate dehydrogenase [EC 1.1.1.42 threo-D-isoci- tide linkages to water did not contribute to the observed trate:NADP+ oxidoreductase (decarboxylating)]. Purified AV* of enzymic reactions. On the other hand, a titration of pig heart IDH was purchased from Calbiochem. The assay AV* by increasing concentrations of salts in the assay solu- solution contained, in a total volume of 5.0 ml, 3.33 mM tion would suggest that water-density-modifying groups on MgCI2, 2.0 mM isocitric acid (Calbiochem, prepared fresh the protein's surface were changing their exposure to water daily). 0.55 mM NADP+, and 25 mM Tris*HCl buffer, pH during catalysis. 8.0 at the assay temperature. Salt concentration and compo- Fig. 2 shows for sition were as indicated in the figures and tables. The reac- that three of the four enzymes studied, tion was initiated by addition of IDH and followed by re- * The volume change accompanying a model solute transfer pro- cording the increase in absorbance at 340 nm as a function cess from hydrocarbon to water equals the difference in the par- of time. tial molar volume of the model solute between the two phases. Downloaded by guest on September 30, 2021 3016 Biochemistry: Low and Somero Proc. Nat. Acad. Sci. USA 72 (1975)

60 80 70 -60 40 0) 50 1.\ E IDH E 40 20 * *PK- 30 LDH /- 20

100 :. 200 300 400- o 10 -A: IKCI, mM ~M~DH' 0 100 200 300 400 -20 Salt, mM FIG. 3. The effect of various cations on the activation volume FIG. 2. The effect of KCl concentration on the activation vol- of the PK reaction: 0, LiCl; *, triethylammonium chloride; 0, umes of the PK, LDH' IDH, and MDH reactions. NaCl; o, CsCl; A, ammonium chloride; A, KCL the neutral salt KCl can effectively titrate a major portion of ty and structure, of a large variety of macromolecular and the activation volume. However, above approximately 300 low-molecular-weight solutes. The effects of these ions on mM concentrations, KCl is unable to further modify AV* solutes have generally been attributed to the different ef- values significantly. This maximum capacity to alter AV* fects of the ions on water structure (23, 24). The cations test- suggests that the volume changes for all transfer processes on ed (Fig. 3), with the exception of Cs+, also follow the Hof- the enzymes' surfaces have been maximally titrated. meister series. Two sets of experiments were performed to determine the A second series of experiments was conducted to deter- mechanism by which salts modify activation volumes. If mine if direct ion binding to polar or charged groups on the salts act by disrupting the dense hydration spheres around proteins' surfaces contributed to the observed ion titration of exposed protein groups, then salts that differ in their effects AtV. The rationale for this investigation is as follows. The on water structure should have differential effects on the withdrawal of an exposed and densely hydrated carboxylate "hydration density" contribution to AV*. group into a protein molecule during catalysis would require Table 2 shows that the relative capacity of various anions the release of a large quantity of electrostricted water, and a to modify the activation volumes of the four enzymic reac- volume increase in the system would occur. However, if the tions generally reflects the position of each anion in the Hof- exposed carboxylate group were neutralized by a cation, the meister series. The Hofmeister series is an empirical ranking amount of electrostricted water that must be released during of ions based on their effects on the properties, e.g., solubili- transfer would ble greatly reduced. Thus, direct ion binding could contribute to the salt titration of AV*. Table 1. Important sources of volume changes Whereas, very weak ion binding is difficult to investigate, in biological processes extremely tight ion binding might be revealed using the fol- Volume lowing approach. Samples of enzyme stock solutions con- change taining 2.2 M ammonium sulfate were treated in two ways. Process (¢m3/mol) One sample was diluted to a final ammonium sulfate con- centration of approximately 3 mM immediately prior to Hydrogen bond formationa -(3-5) assay. A second sample was exhaustively dialyzed to remove Hydrophobic interactions all ammonium sulfate. If ions remained bound to the freshly CH4 in hexane -+ CH in waterb -22.7 diluted enzyme but were completely removed from the di- C0H6 (pure liquid) -+C,0H6 in waterb -6.2 alyzed enzyme, differences in the salt titration curves of the H20 (pure) -+ H20 (dilute) in 1,1,1-tri- chloroethaneC +4.3 two enzyme samples might occur. H20 (pure) -. H20 (dilute) in CC014C +13.6 No differences were noted between diluted and dialyzed Ionic interactions samples of LDH, MDH, and IDH. However, for PK (Fig. 4), Lysine (neutral) -+ lysine (+)a -26.4 Table 2. Effects of various salts on AV* values Acetic acid - acetate -+ H+a -11.5 (in cm3/mol) Glutamic acid -+ glutamate- + H+a -12.7 Mg-ATP complex -> ATP + Mg++d -22 Salta MDHb IDHb LDHC PKb Change in exposure of polar group Methanol in CC001- methanol in H2Oe -7.1 KF -27 32 -11 - . -13 23 -2 7 Ethanol in CC14 - ethanol in H2Oe -4.9 . K2SO4 n-Propanol in CC14 -, n-propanol in H2Oe -6.4 KCl -2 27 6 14 -- 3@KBr 0 29 13 25 Methanol (pure) methanol in H2Of -2.44 0 Ethanol (pure) -> ethanol in H20f -3.4 KI 20 39 16 37 n-Propanol (pure) nepropanol in H20f -4.52 KSCN 23 38 20 54 Helix-coil transitiong +1 a The salts are listed according to common "Hofmeister series" ranking (23). a Suzuki and Taniguchi (15); bKauzmann (5); CMasterton and b The effects of 200 mM salt concentrations were compared for this Seiler (16); d Rainford et al. (17); e values calculated from data enzyme. given by Duboc (18) and Friedman and Scheraga (8); f Friedman c The effects of 400 mM salt concentrations were compared for this. and Scheraga (8); g Noguchi (19). enzyme. Downloaded by guest on September 30, 2021 Biochemistry: Low and Somero Proc. Nat. Acad. Sci. USA 72 (1975) 3017

350t IDH 30r~~~~ X 25 O -0 hi0 25 E E o LI *4. 'ci 55 0 100 200 300 400 CsCI, mM 0 , a* * * LDH 80 .2 .4 .6 .8 1.0 1.2 n-propanol, Molarity 70 FIG. 5. The effect of n-propanol on the activation Volumes of B the and LDH reactions. 60 PK, IDH, volumes to considerable degrees, for some enzymes a portion 0 50 of AV* remains unaltered in the presence of even very high E salt concentrations (Figs. 2-4). We believe that this nontitra- E 40 table fraction of AV* represents what we have termed a *30 * "structural" volume change, i.e., a change in the volume oc- cupied by the protein molecule itself, exclusive of any hy- 20 dration sphere. (The size of the "structural" volume change therefore can be taken as a weak measure of the magnitude 16 of the conformational change that occurs during catalysis.) Since protein thermal stability and, by extension, confor- 0 100 200 300 400 mational flexibility, are related to the adaptation tempera- LiCI, mM ture of a protein (27, 28), we reasoned that the nontitratable FIG. 4. The effects of CsCl (A) and LiCl (B) on the activation fraction of AV*, if it represents a "structural" volume volume of the PK reaction. Open symbols are data obtained using change, might differ among homologues of an enzyme from enzyme that was diluted to a final ammonium sulfate concentra- organisms adapted to different temperatures. This expecta- tion of approximately 3 mM immediately prior to assay. Closed tion is realized (Fig. 6). Similar correlations between the symbols are for assays performed with exhaustively dialyzed en- zyme. 40p- *CHICKEN the dialyzed samples showed a much stronger titration effect RABBIT @ at low salt concentrations. These differences can be rational- ized most easily on the basis of the finding that specific cat- ion (K+, NH4+) binding sites exist on PK (25). These sites may be vacant on the dialyzed enzyme but partially filled 30- on the diluted enzyme. No specific ion binding sites, or spe- cific ion requirements, are known for the three dehydroge- SKIPJACK TUNA * Red Muscle nases. 0 We also determined the effect of a nonionic solute, n-pro- QCa

panol, on the activation volumes of three of the enzymic 0 Mugil cpholus* / Bufo marinus reactions (Fig. 5). Only very small effects were noted, even / SKIPJACK TUNA 0 * White Muscle when n-propanol concentrations were raised to levels suffi- CI- 2 Heart cient to denature the enzymes (approximately 1 M and above). From all of the above solute studies, we conclude that the lo k Scorpoena outotta movement of water-density-modifying protein groups at the Cancer mogister enzyme water interface contributes significantly to activa- tion volumes. Support for this conclusion is provided by data on the three-dimensional structures of enzymes. X-ray crys- tallography of chymotrypsin (26) has revealed that 393 un- __ charged but polar nitrogen and oxygen atoms, 34 ionized 5 K 15 20 25 30 carboxylate and amino groups, and 26 hydrophobic side- S Trematomus borchgrevinki chains lie at the protein-water interface. Additional water- AV* (cm3/mole) density-modifying groups are only partially exposed to water. It FIG. 6. The relationship between the normal cell temperature would, therefore, be very difficult for a conforma- of a species and the size of the nontitratable ("structural") activa- tional change to occur without a concomitant change in the tion volume for the PK reaction. Mugil cephalus, Scorpaena gu- protein's hydration density. tatta, and Trematomus borchgrevinki are teleost fishes; Cancer Finally, in spite of the fact that salts can titrate activation magister is a crustacean; and Bufo marinus is a toad. Downloaded by guest on September 30, 2021 3018 Biochemistry: Low and Somero Proc. Nat. Acad. Sci. USA 72 (1975)

nontitratable fraction of AVt and adaptation temperature 8. Friedman, M. & Sheraga, H. A. (1965) J. Phys. Chem. 69, were found for the dehydrogenases; these relationships will 3795-3800. be discussed more fully elsewhere. We regard the correla- 9. Boje, L. & Hvidt, A. (1972) Blopolymers 11, 2357-2364. tion between the adaptation temperature of the enzyme and 10. Low, P. S. & Somero, G. N. (1975) Proc. Nat. Acad. Scd. USA, the size of the nontitratable fraction of AV* as support for in press. 11. Laidler, K. J. (1950) Arch. Blochem. 30,226-236. our contention that the nontitratable fraction of AV* is, in 12. Dowd, J. & Riggs, D. (1965) J. Biol. Chem. 249,863-869. fact, indicative of a change in the volume of the protein it- 13. Mustafa, T., Moon, T. W. & Hochachka, P. W. (1971) Am. self. Zool. 11, 451-466. 14. Bondar, R. J. L. & Pon, N. G. (1969) Biochim. Biophys. Acta 191,743-747. We thank Dr. Peter W. Hochachka for generous use of his high 15. Suzuki, K. & Taniguchi, Y. (1972) in The Effects of Pressure pressure spectrophotometer. We thank Drs. Daniel Atkinson, Jef- on Living Organisms, eds. Sleigh, M. A. & MacDonald, A. G. frey Bada, Joris Gieskes, Stanley Miller, and Rufus Lumry for their (Academic Press, New York), pp. 103-124. helpful suggestions in the preparation of this manuscript. This work 16. Masterton, W. L. & Seiler, H. K. (1968) J. Phys. Chem. 72, was supported by NSF Grant BMS 74-1735. 4257-4262. 17. Rainford, P., Noguchi, H. & Morales, M. (1965) Biochemistry 4, 1958-1965. 1. Low, P. S. & Somero, G. N. (1975) Comp. Biochem. Physiol., 18. Duboc, C. (1969) Bull. Soc. Chim. Fr. 7,2260-2270. in press. 19. Noguchi, H. (1966) Blopolymers 4,1105-1113. 2. Johnson, F. H. & Eyring, H. (1970) in High Pressure Effects 20. Wirth, H. E. (1948) J. Am. Chem. Soc. 70,462-465. on Cellular Processes, ed. Zimmerman, A. M. (Academic 21. Ward, G. K. & Millero, F. J. (1974) J. Solution Chem. 3, Press, New York), pp. 1-44. 431-444. 3. Hochachka, P. W. & Somero, G. N. (1973) Strategies of Bio- 22. Millero, F. J. (1969) J. Phys. Chem. 73,2417-2420. chemical Adaptation (W. B. Saunders Co., Philadelphia), 358 23. von Hippel, P. H. & Schleich, T. (1969) in Structure and Sta- PP. bility of Biological Macromolecules, eds. Timesheff, S. N. & 4. Brandts, J. F., Oliveira, R. J. & Westort, C. (1970) Biochemis- Fasman, G. D. (Marcel Dekker, Inc., New York), pp. 417-574. try 9, 1038-1047. 24. Klotz, I. M. (1965) Fed. Proc. 24 (Suppl. 15), 5-24. 5. Kauzmann, W. (1059) Adv. Protein Chem. 14,1-63. 25. Nowak, T. (1973) J. Biol. Chem. 248,7191-7196. 6. Zipp, A. & Kauzmann, W. (1973) Biochemistry 12, 4217- 26. Birktoft, J. J. & Blow, D. M. (1972) J. Mol. Biol. 68,187-240. 4228. 27. Somero, G. N. (1975) J. Exp. Zool., in press. 7. Nemethy, G. & Sheraga, H. A. (1962) J. Phys. Chem. 66, 28. Singleton, R. & Amelunxen, R. (1973) Bactertol. Rev. 37, 1773-1789. 320-342. Downloaded by guest on September 30, 2021