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Specific and non-specific binding of long-chain fatty acids to firefly luciferase: cutoff at octanoate
Biochimica et Biophysica Acta 1426 (1999) 143^150
Speci¢c and non-speci¢c binding of long-chain fatty acids to ¢re£y luciferase: cuto¡ at octanoate Hitoshi Matsuki 1 , Atsuya Suzuki 2 , Hiroshi Kamaya, Issaku Ueda * Department of Anesthesia, Anesthesia Service 112A, DVA Medical Center, and University of Utah School of Medicine, 500 Foothill Blvd., Salt Lake City, UT 84148, USA Received 8 August 1998; received in revised form 19 October 1998; accepted 19 October 1998
Abstract Firefly luciferase emits a burst of light when the substrates luciferin and ATP are mixed in the presence of oxygen. We (I. Ueda, A. Suzuki, Biophys. J. 75 (1998) 1052^1057) reported that long-chain fatty acids are specific inhibitors of firefly luciferase in competition with luciferin in WM ranges. They increased the thermal transition temperature. In contrast, 1-alkanols of the same carbon chain length inhibited the enzyme non-competitively in mM ranges and decreased the transition temperature. The present study showed that the action of fatty acids switched from specific to non-specific when the carbon chain length was reduced below C8 (octanoate). The fatty acids longer than C10 inhibited the enzyme in WM ranges whereas those shorter than C8 required mM ranges to inhibit it. The longer fatty acids increased whereas shorter fatty acids decreased the transition temperature. The Hill coefficients of longer chain bindings were less than one whereas those of shorter chain were more than one. The shorter fatty acids interacted with the enzyme cooperatively at multiple sites. Binding of the longer fatty acids is limited. Fatty acids longer than C10 are high-affinity specific binders and followed Koshland's induced-fit model. Those shorter than C8 are low-affinity non-specific denaturants and followed Eyring's rate process model. These results contradict the general consensus that the size of the receptor cavity discriminates specific binders. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Protein-fatty acids interaction; Protein-anesthetic interaction ; Fire£y luciferase; Rapid reaction kinetics; Irreversible phase transition
1. Introduction When ¢re£y luciferase is mixed with ATP and luciferin in the presence of oxygen, a £ash of light * Corresponding author. Fax: +1 (801) 5842545; E-mail: [email protected] 1 Present address: Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, Minamijosanjima, Tokushima 770, Japan. 2 Present address: Department of Neurology, Chiba University School of Medicine, Chuoku, Chiba 260-0856, Japan.
appears after 25 ms of complete darkness, then decays to a low grade light intensity. The low light intensity lasts for several hours [1^3]. E Luciferin ATP3EWLuciferylAMP PPi EWLuciferylAMP O2 ! EWOxyluciferylAMP CO2 Light EWOxyluciferylAMP ! E Oxyluciferin AMP where E is ¢re£y luciferase and PPi is pyrophos-
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 8 ) 0 0 1 4 8 - 2
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phate. Because the initial £ash intensity is a transient event, it is not in a steady-state condition. Enzyme kinetics must be analyzed under a steady state. Several methods have been used to obtain steady-state reaction rates. These include scintillation counting, the slope of the linear part of the integral of the light output, etc. The initial reaction is freely reversible because the energy level does not change appreciably; one high-energy bond (between AMP and PPi ) is broken and one high-energy bond is made (lucyferylAMP). When an adequate amount of pyrophosphate is added, the initial £ash disappears, and the steady-state light intensity is observed for the time period proportional to the added amount of pyrophosphate [1,4]. Under the pyrophosphate-induced steady-state condition, we [5^7] reported that long-chain fatty acids inhibited ¢re£y luciferase in WM ranges in competition with the substrate luciferin, whereas alcohols with comparable carbon chain length [7] and volatile anesthetics [6] inhibited the enzyme in mM ranges non-competitively with luciferin. By high-pressure stopped-£ow, we [8] established according to the rapid reaction kinetic theory [9] that the initial peak intensity represents the total active enzyme concentration (Etotal = Efree +ES+ESP) and is unrelated to the reaction kinetics. The report by Franks and Lieb [10] that anesthetics and alcohols competed with luciferin was in error because the analysis was based on the initial £ash intensity. By di¡erential scanning calorimetry (DSC) we [5^7] showed that thermal denaturation of ¢re£y luciferase occurred at about 39³C with the transition enthalpy, vHcal , of 92 kcal mol31 . Alcohols and anesthetics decreased whereas long-chain fatty acids increased the transition temperature. The cooling scan, however, did not show the endothermic peak. In this context, the transition was irreversible. Thermal transitions of most multi-domain large proteins are irreversible. Kinetic theories have been proposed [11^15] to analyze the irreversible transition in large proteins according to the Lumry-Eyring model [15].
analysis measures the temperature dependence of the irreversibly denatured fraction. It is often argued that irreversible denaturation at high temperatures is irrelevant to the biological actions at physiological temperatures. The ¢rst-order transition ensues after multiple reversible intermediate states. The shift of the transition temperature is a quantitative parameter for the vulnerability of a protein for the reversible transitions at lower temperatures. By Fourier transform infrared (FTIR) spectroscopy we [5] showed the presence of reversible multiple intermediate states in ¢re£y luciferase before irreversible denaturation. The Lorentzian deconvolution of the Amide-IP peak showed an increase of the L-structure content in the expense of the K-helix content when heated or when anesthetics or alcohols were added. Ethanol induced the K-to-L uncoiling even at 5³C, much below the denaturation temperature [5]. In contrast, myristate and other luciferin competitors [5] protected the enzyme from thermal denaturation. A similar change in the secondary structure was seen in a simpler polypeptide, poly(L-lysine) [16]. FTIR and the mean residue ellipticity of circular dichroism showed that addition of ethanol increased the L-structure and decreased the K-helix content. During the study, we noticed that the inhibitory potency of fatty acids on ¢re£y luciferase suddenly decreased at the carbon chain length about eight. The inhibitor concentration that decreased the light intensity 50% (IC50 ) of short-chain fatty acids jumped from WM to mM ranges. Short-chain fatty acids decreased the thermal transition temperature. The mode became identical to that of anesthetics and alcohols. The present study compared the e¡ects of butyrate (C4), hexanoate (C6), and octanoate (C8) with those of the longer chain fatty acids on the thermal denaturation temperature and the steadystate luminescence intensity of ¢re£y luciferase. Alcohols also lose the inhibitory potency at carbon chain length about 8^10, which is known as the cuto¡ point. In the alcohol series, however, it is the longer alcohols that lose their potency.
N3I1 3I2 3 ::: 3In 3U ! D where N is the native enzyme, I1 , I2 ... In are the intermediate states, U is the reversibly unfolded state and D is the irreversibly denatured state. Kinetic
2. Method Lyophilized crystalline ¢re£y luciferase from Pho-
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tinus pyralis, sodium salt of D-luciferin, glycylglycine, ATP, and the sodium salts of fatty acids between butyrate (C4) and octadecanoate (C18), were obtained from Sigma (St. Louis, MO). 2.1. Bioluminescence Fire£y luciferase and luciferin were dissolved in 100 mM glycylglycine bu¡er pH 7.8 and placed in a 1.0 ml cuvette. Na2 ATP with MgSO4 solution was rapidly injected into the ¢re£y luciferase solution. The ¢nal concentrations were ¢re£y luciferase 20 Wg ml31 , luciferin 100 WM, ATP 5.0 mM, and MgSO4 10 mM in a total 600 Wl. The fatty acids were added in both solutions. The light output was measured by a Hitachi-Perkin Elmer 139 Spectrophotometer (Norwalk, CT). The photomultiplier output was monitored by a Nicolet 310 Digital Recording Oscilloscope (Madison, WI) and downloaded on £oppy disks. The light output was integrated by software ORIGN (MicroCal, Northampton, MA). 2.2. Thermal transition The total heat absorbed equals the calorimetrically obtained denaturation enthalpy, vHcal . However, irreversibility distorts the DSC thermogram. Therefore, the denaturation temperature was estimated at the temperature where the irreversible denaturation is half completed, T1=2 . Because irreversible denaturation is kinetically controlled, T1=2 varies by the
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scan rate. The thermogram shifts to lower temperatures when the protein concentration is higher or the scan rate is slower [5^7]. We found that the ¢re£y luciferase solution suddenly becomes turbid at the thermal denaturation temperature [6,7]. We compared the turbidity data with those of DSC, obtained by a MicroCal MC2 di¡erential scanning calorimeter (Northampton, MA) at the same scan rate of 1.0³C min31 and the same ¢re£y concentration of 3.0 mg ml31 in 0.5 M glycylglycine bu¡er pH 7.8. The ¢re£y luciferase concentration in the DSC study was higher than in the light intensity study, because the DSC study requires higher protein concentrations to obtain reproducible results [6,7]. When measured by the turbidity, tracings of various scanning rates were parallel to each other [6]. The di¡erential of the turbidity scan was almost the same as the DSC thermogram (Fig. 1). The peak was at 38.1³C with a half-height width of 3.5³C. The scanning rate did not a¡ect the halfheight width of the transition. For a reversible transition, the slowest scan rate has been chosen to maintain a quasi-equilibrium condition during the ¢rstorder transition. However, no matter how slow the scan rate is maintained, or how small the sample size is reduced, one cannot maintain a thermodynamically perfect equilibrium condition represented by zero half-height temperature width. Nevertheless this does not mean that the transition is second order. With irreversible transition, some investigators [11,13] suggested to use a faster scan rate on the ground that the irreversible step occurs at temper-
Table 1 E¡ects of fatty acids on the IC50 , Hill number (nH ), the concentrations that decreased (9 C8) or increased (v C10) phase transition temperature (vT1=2 ) by 1.0³C, and the logarithm of octanol/water partition coe¤cients, log P C4 C6 C8 C10 C12 C14 C16 C18
IC50
naH
vTb1=2
13.6 mM 3.4 mM 2.9 mM 13.2 WM 1.2 WM 0.68 WM 0.67 WM 0.63 WM
3.02 1.04 0.81 0.83 0.89 0.89 0.95 0.95
10.34 mM 3.28 mM 2.46 mM 7.4 WM 2.3 WM 1.2 WM 1.0 WM 0.87 WM
a
log Pc 32.70 31.70 30.70 0.30 1.30 2.31 3.31 4.31
Hill number. Ligand concentrations that changed the transition temperature 1.0³C. Fatty acids with carbon chain lengths shorter than C8 decreased and those longer than C10 increased the T1=2 . c Hansch and Clayton [27]. b
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atures above the reversible transition. It is expected that when the scan rate is faster, there may be less chance of the irreversible change a¡ecting the reversible process. Whatever the rationale, our study showed that the half-height temperature width of the transition did not vary by a scan rate between 2.0 and 0.1³C min31 . The e¡ects of modi¢ers on the denaturation temperature can be meaningfully compared when the scan rates and protein concentrations are the same. The transition temperature, T1=2 , was measured by the turbidity with the solution containing ¢re£y luciferase 0.8 mg ml31 in 100 mM glycylglycine bu¡er pH 7.8. The heating scan rate was 1.0³C min31 . The transmittance was measured at 400 nm in a 1.0 cm light path cuvette by a Perkin Elmer 554 Spectrophotometer (Norwalk, CT) equipped with a programmable digital linear temperature controller and a Peltier heat exchanger. The actual temperature in the sample cuvette was measured by a ¢lament thermistor (YSI, Yellow Springs, OH) inserted into the cuvette and a DigiTech Model 5810 digital thermometer (United Systems, Dayton, OH) with 0.01³C resolution. The light output and the temperature were monitored by the digital recording oscilloscope and downloaded on £oppy disks.
Fig. 1. Comparison of the di¡erential scanning calorimetry thermogram with the temperature scan of spectrophotometry. The ¢re£y luciferase concentration was 3.0 mg ml31 in 0.5 M glycylglycine bu¡er pH 7.8 and the scan rate was 1.0³C min31 . Continuous line: DSC thermogram; broken line: di¡erential of the transmittance at 400 nm.
Fig. 2. IC50 values of fatty acids on the steady-state light intensity of ¢re£y luciferase. Ordinate: the logarithm of the Wmolar concentrations of fatty acids; abscissa: the number of carbon atoms.
3. Results 3.1. Light intensity The steady-state light intensity was estimated from the slope of the linear part of the integral of the light output. The IC50 values decreased with the elongation of the carbon chain: C16: 0.67 WM, C14: 0.68 WM, C12: 11.2 WM, C10: 13.2 WM, C8: 2.9 mM, C6: 3.4 mM, and C4: 13.6 mM (Fig. 2, Table 1). There is a sudden discontinuous change from WM to mM at C10-C8. The IC50 values changed from 13.2 WM of C10 to 2.9 mM of C8, a 220-fold jump in IC50 . In contrast, the change in IC50 from C12 to C10 was only 11-fold. The dose-response curves of the three fatty acids above C8 are shown in Fig. 3. The dose-response curves are not parallel. This is unusual because the dose-response curves of similar compounds such as 1-alkanol series are parallel. The dose-response curves of the fatty acids longer than C10 were parallel [6,7]. By assuming n molecules of the ligand L bind ¢re£y luciferase E with association constant K, the interaction is written K
E nL 3 ELn and
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K
ELn ELn
1
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Fig. 3. Dose-response curves of C8, C6, and C4 on light intensity. Ordinate: the ratio of the steady-state light intensity with and without the inhibitors; abscissa: the logarithm of the molar concentrations of inhibitors. Triangles, C8; squares, C6; circles, C4. Each point is the average of ¢ve determinations and standard errors are inside of the symbols.
ELn KL E n
2
The total enzyme concentration is E0 E ELn
3
Then KLn
ELn E0 3ELn
4
Fig. 5. Dose-dependent e¡ect of C8 on the denaturation temperature. Ordinate: the transmittance of the ¢re£y luciferase solution at 400 nm; abscissa: the temperature of the solution. The scan rate was 1.0³C min31 . The C8 concentrations are from the left: control (thick line), 0.25, 1.0, 4.19, 9.98, and 19.98 mM.
where [E0 ]3[ELn ] is the light intensity in the presence of inhibitor. [ELn ] is the portion where the light intensity is inhibited. We designate the control light intensity without the inhibitor by I1 , and the light intensity with the inhibitor by I2 . Then, [ELn ] = I1 3I2 and [E0 ]3[ELn ] = I2 , and Eq. 4 becomes I 1 3I 2 KLn I2 By taking the logarithm, I1 ln 31 n lnL3ln K I2
Fig. 4. Hill plot. The inhibitor e¡ects are plotted according to Eq. 6 in the text. Ordinate: the logarithm of (I1 /I2 31) where I1 and I2 are the light intensities of ¢re£y luciferase in the absence and presence of the inhibitors, respectively; abscissa : the logarithm of the molar concentrations of the inhibitors. Triangles, C8; squares, C6; circles, C4.
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5
6
At constant temperature and pressure, ln K becomes constant. By plotting the left-hand side to the ligand concentrations, a straight line is obtained. The slope of the straight line indicates the Hill number, nH , of ligand molecules that bind to ¢re£y luciferase. The sigmoidal dose-response curves were linearized according to the above equation and are shown in Fig. 4. The Hill numbers were: C8: 0.812, C6: 1.038, and C4: 3.017. The nH values of the fatty acids longer than C8 were less than one (Table 1). 3.2. Thermal transition temperature The fatty acids with longer carbon chain (v C8)
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Fig. 6. Dose-dependent e¡ect of C6 on the denaturation temperature. The C6 concentrations are from the left: 14.97, 10.14, 5.06, 2.54, 1.27 mM and control (thick line).
increased the thermal denaturation temperature. In contrast, shorter chain fatty acids (9 C6) decreased it. Figs. 5 and 6 show the e¡ects of C8 and C6 on the temperature scan, respectively. The dose-dependent e¡ects of C8, C6, and C4 on the thermal denaturation temperature are shown in Fig. 7. C8 increased T1=2 by 1.0³C at 2.46 mM. C6 and C4 decreased T1=2 by 1.0³C at 3.28 and 10.34 mM, respectively. The fatty acids longer than C10 increased T1=2 . The concentrations that increased T1=2 by 1.0³C were: C10: 7.4 WM, C12: 2.3 WM, C14: 1.2 WM, C16: 1.0 WM, and C18: 0.87 WM (Fig. 8, Table 1).
Fig. 7. Dose-dependent e¡ects of the short-chain fatty acids on the denaturation temperature. Ordinate: change of the denaturation temperature in ³C; abscissa: solution temperature. Triangles, C8; squares, C6; circles, C4. C8 increased, whereas C6 and C4 decreased the denaturation temperature.
Fig. 8. Concentrations of the inhibitor that changed the denaturation temperature T1=2 , by 1.0³C. Ordinate: log concentrations of the inhibitor in Wmolar that changed T1=2 by 1.0³C; abscissa: the number of carbon atoms in the fatty acids. Open circles are the fatty acids hat increased T1=2 . Closed circles are those that decreased T1=2 .
4. Discussion We [5^7] have shown that long-chain fatty acids are strong speci¢c inhibitors of ¢re£y luciferase in competition with luciferin. The initial step of the light emission involves activation of luciferin by ATP to form acyl-AMP between the carboxyl moiety of luciferin and phosphate moiety of AMP. Luciferin is a heterocyclic carboxylate. The initial step of oxidation of fatty acids by acyl-CoA transferases also involves activation of fatty acids by ATP to form acyl-AMP. The homology between the two has been reported [17^19]. It is not surprising that fatty acids compete with luciferin. There is no structural similarity between luciferin and anesthetics. It would be surprising if anesthetics competed with luciferin. To our knowledge, all reports on anesthetic e¡ects on enzymes concluded allosteric interaction. The only exception was by Franks and Lieb [10] who reported that anesthetics compete with luciferin by the use of Lineweaver-Burk plots on the initial £ash intensity in ¢re£y luciferase. Aside from the use of the non-steady-state condition in analyzing the inhibition kinetics, the use of the Lineweaver-Burk plot in multiple-stage reactions requires justi¢cation for the applicability of the linearization procedure. With ¢re£y luciferase, the released free enzyme E and ¢nal
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product oxyluciferin react in the presence of ATP [2,20]. E Oxyluciferin ATP3EWOxylucyferylAMP PPi The multiplicity of the reaction of ¢re£y luciferase invalidates the applicability of the Lineweaver-Burk plot that deals with one substrate and one inhibitor. The above equation shows that oxyluciferin becomes an additional inhibitor. Even if the plot shows apparent competition, the exact binding mode may not be established in this enzyme reaction with two inhibitors and three substrates. The present study showed that there are two modes in the action of fatty acids on ¢re£y luciferase. The action mode changed at the carbon chain length about C8. The IC50 increased from 13.2 WM of C10 to 2.9 mM of C8, a 220-fold jump. The change from C12 to C10 was 11-fold. A more dramatic change was observed with the transition temperature, where those longer than C10 increased the transition temperature, whereas those shorter than C8 decreased the transition temperature. The concentration that changed the transition temperature 1.0³C (negative or positive) increased from 7.4 WM of C10 to 2.46 mM of C8, a 332-fold jump. The change from C12 to C10 was 3.2-fold. The dose-response curves showed a large change as shown in Fig. 3. The Hill numbers changed from less than one at C8 to more than one at C6. The Hill number assumes a highly cooperative interaction between enzyme E and n molecules of ligand L to yield only one ELn molecule. The intermediates, EL1 , EL2 ... ELn31 are ignored. Therefore nH s 1 means that at least two or more ligand molecules are cooperatively bound to the enzyme. Conversely, nH 91 means noncooperative limited binding. The real binding number must be estimated by the Scatchard plot. It is possible that the actual binding numbers of the ligand with nH s 1 are much more than the Hill number. We reported by 19 F-NMR [21] that the maximum binding number, Bmax , of halothane to bovine serum albumin (BSA) was 34, and by di¡erential titration calorimetry [22] that the Bmax of chloroform, bound to BSA, was 13.2. Both values were obtained by the Scatchard plot. The present result shows that the binding number suddenly increased when the carbon chain length becomes shorter than
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C8. The binding mode changed from a non-cooperative limited number of binding sites to a highly cooperative multiple binding sites. Koshland [23,24] proposed the induced-¢t model where the ligands that bind to the substrate binding sites induce the protein structure to a high-energy native state. Eyring [25,26] proposed the rate process model where anesthetics and alcohols bind non-speci¢cally to proteins and reversibly unfold the protein into a less active state by releasing the surface-bound water molecules. Fatty acids longer than C10 are speci¢c binders to the high-energy native state of ¢re£y luciferase. Short-chain fatty acids are non-speci¢c binders to the reversibly unfolded enzyme. The sign of the logarithm of the oil/water partition coe¤cients (log P) of the short-chain fatty acids changes from positive (C10) to negative (C8) at the identical position where the change from the speci¢c to non-speci¢c interaction occurred. The negative sign signi¢es that the short-chain fatty acids distribute more to the aqueous phase than to the organic phase (oil/water 6 1). Due to the strong hydrophobicity of the luciferin binding site [2], shorter fatty acids are apparently excluded from the luciferin binding site. Acknowledgements Supported by DVA Medical Research Funds.
References [1] W.D. McElroy, H.H. Seliger, Fed. Proc. 21 (1962) 1006^ 1012. [2] M. DeLuca, W.D. McElroy, Biochemistry 13 (1974) 921^ 925. [3] M. DeLuca, W.D. McElroy, Methods Enzymol. 57 (1978) 3^ 15. [4] I. Ueda, F. Shinoda, H. Kamaya, Biophys. J. 66 (1994) 2107^2110. [5] J.S. Chiou, I. Ueda, J. Pharm. Biomed. Anal. 12 (1994) 969^ 975. [6] I. Ueda, A. Suzuki, Biochim. Biophys. Acta 1380 (1998) 313^319. [7] I. Ueda, A. Suzuki, Biophys. J. 75 (1998) 1052^1057. [8] I. Ueda, H. Minami, H. Matsuki, T. Inoue, Biophys. J. in press.
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[9] K. Hiromi, Kinetics of Fast Enzyme Reactions, John Wiley and Sons, New York, 1979, pp. 226^232. [10] N.P. Franks, W.R. Lieb, Nature 310 (1984) 599^601. [11] J.M. Sa¨nchez-Ruiz, J.L. Lo¨pez-Lacomba, M. Cortijo, P.L. Mateo, Biochemistry 27 (1988) 1648^1652. [12] J.R. Lepock, K.P. Ritchie, M.C. Kolios, A.M. Rodahl, K.A. Heinz, J. Kruuv, Biochemistry 31 (1992) 12706^12712. [13] A. Herna¨ndez-Arana, A. Rojo-Dom|¨nguez, M.M. Altamirano, M.L. Calcagno, Biochemistry 32 (1993) 3644^3648. [14] V.L. Shnyrov, M.J. Marcos, E. Villar, Biochem. Mol. Biol. Int. 4 (1996) 647^656. [15] R. Lumry, H. Eyring, J. Phys. Chem. 58 (1954) 110^120. [16] A. Shibata, M. Yamamoto, T. Yamashita, J.S. Chiou, H. Kamaya, I. Ueda, Biochemistry 31 (1992) 5728^5733. [17] H. Suzuki, Y. Kawarabayashi, J. Kondo, T. Abe, K. Nishikawa, S. Kimura, T. Hashimoto, T. Yamamoto, J. Biol. Chem. 265 (1990) 8681^8685.
[18] P.C. Babbitt, G.L. Kenyon, B.M. Martin, H. Charest, M. Slyvestre, J.D. Scholten, K.H. Chang, P.H. Liang, D. Dunaway-Mariano, Biochemistry 31 (1992) 5594^5604. [19] L. Ye, L.M. Buck, H.L. Schae¡er, F.R. Leach, Biochim. Biophys. Acta 1339 (1997) 39^52. [20] R. Lee, W.D. McElroy, Biochemistry 8 (1969) 130^136. [21] T. Yoshida, M. Tanaka, Y. Mori, I. Ueda, Biochim. Biophys. Acta 1334 (1997) 117^122. [22] I. Ueda, M. Yamanaka, Biophys. J. 72 (1997) 1812^1817. [23] D.E. Koshland Jr., Proc. Natl. Acad. Sci. USA 44 (1958) 98^104. [24] D.E. Koshland Jr., Science 142 (1963) 1533^1541. [25] H. Eyring, J.L. Magee, J. Cell Comp. Physiol. 20 (1942) 169^177. [26] H. Eyring, Science 154 (1966) 1609^1613. [27] C. Hansch, J.M. Clayton, J. Pharm. Sci. 62 (1973) 1^21.
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Speci¢c and non-speci¢c binding of long-chain fatty acids to ¢re£y luciferase: cuto¡ at octanoate Hitoshi Matsuki 1 , Atsuya Suzuki 2 , Hiroshi Kamaya, Issaku Ueda * Department of Anesthesia, Anesthesia Service 112A, DVA Medical Center, and University of Utah School of Medicine, 500 Foothill Blvd., Salt Lake City, UT 84148, USA Received 8 August 1998; received in revised form 19 October 1998; accepted 19 October 1998
Abstract Firefly luciferase emits a burst of light when the substrates luciferin and ATP are mixed in the presence of oxygen. We (I. Ueda, A. Suzuki, Biophys. J. 75 (1998) 1052^1057) reported that long-chain fatty acids are specific inhibitors of firefly luciferase in competition with luciferin in WM ranges. They increased the thermal transition temperature. In contrast, 1-alkanols of the same carbon chain length inhibited the enzyme non-competitively in mM ranges and decreased the transition temperature. The present study showed that the action of fatty acids switched from specific to non-specific when the carbon chain length was reduced below C8 (octanoate). The fatty acids longer than C10 inhibited the enzyme in WM ranges whereas those shorter than C8 required mM ranges to inhibit it. The longer fatty acids increased whereas shorter fatty acids decreased the transition temperature. The Hill coefficients of longer chain bindings were less than one whereas those of shorter chain were more than one. The shorter fatty acids interacted with the enzyme cooperatively at multiple sites. Binding of the longer fatty acids is limited. Fatty acids longer than C10 are high-affinity specific binders and followed Koshland's induced-fit model. Those shorter than C8 are low-affinity non-specific denaturants and followed Eyring's rate process model. These results contradict the general consensus that the size of the receptor cavity discriminates specific binders. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Protein-fatty acids interaction; Protein-anesthetic interaction ; Fire£y luciferase; Rapid reaction kinetics; Irreversible phase transition
1. Introduction When ¢re£y luciferase is mixed with ATP and luciferin in the presence of oxygen, a £ash of light * Corresponding author. Fax: +1 (801) 5842545; E-mail: [email protected] 1 Present address: Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, Minamijosanjima, Tokushima 770, Japan. 2 Present address: Department of Neurology, Chiba University School of Medicine, Chuoku, Chiba 260-0856, Japan.
appears after 25 ms of complete darkness, then decays to a low grade light intensity. The low light intensity lasts for several hours [1^3]. E Luciferin ATP3EWLuciferylAMP PPi EWLuciferylAMP O2 ! EWOxyluciferylAMP CO2 Light EWOxyluciferylAMP ! E Oxyluciferin AMP where E is ¢re£y luciferase and PPi is pyrophos-
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 8 ) 0 0 1 4 8 - 2
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phate. Because the initial £ash intensity is a transient event, it is not in a steady-state condition. Enzyme kinetics must be analyzed under a steady state. Several methods have been used to obtain steady-state reaction rates. These include scintillation counting, the slope of the linear part of the integral of the light output, etc. The initial reaction is freely reversible because the energy level does not change appreciably; one high-energy bond (between AMP and PPi ) is broken and one high-energy bond is made (lucyferylAMP). When an adequate amount of pyrophosphate is added, the initial £ash disappears, and the steady-state light intensity is observed for the time period proportional to the added amount of pyrophosphate [1,4]. Under the pyrophosphate-induced steady-state condition, we [5^7] reported that long-chain fatty acids inhibited ¢re£y luciferase in WM ranges in competition with the substrate luciferin, whereas alcohols with comparable carbon chain length [7] and volatile anesthetics [6] inhibited the enzyme in mM ranges non-competitively with luciferin. By high-pressure stopped-£ow, we [8] established according to the rapid reaction kinetic theory [9] that the initial peak intensity represents the total active enzyme concentration (Etotal = Efree +ES+ESP) and is unrelated to the reaction kinetics. The report by Franks and Lieb [10] that anesthetics and alcohols competed with luciferin was in error because the analysis was based on the initial £ash intensity. By di¡erential scanning calorimetry (DSC) we [5^7] showed that thermal denaturation of ¢re£y luciferase occurred at about 39³C with the transition enthalpy, vHcal , of 92 kcal mol31 . Alcohols and anesthetics decreased whereas long-chain fatty acids increased the transition temperature. The cooling scan, however, did not show the endothermic peak. In this context, the transition was irreversible. Thermal transitions of most multi-domain large proteins are irreversible. Kinetic theories have been proposed [11^15] to analyze the irreversible transition in large proteins according to the Lumry-Eyring model [15].
analysis measures the temperature dependence of the irreversibly denatured fraction. It is often argued that irreversible denaturation at high temperatures is irrelevant to the biological actions at physiological temperatures. The ¢rst-order transition ensues after multiple reversible intermediate states. The shift of the transition temperature is a quantitative parameter for the vulnerability of a protein for the reversible transitions at lower temperatures. By Fourier transform infrared (FTIR) spectroscopy we [5] showed the presence of reversible multiple intermediate states in ¢re£y luciferase before irreversible denaturation. The Lorentzian deconvolution of the Amide-IP peak showed an increase of the L-structure content in the expense of the K-helix content when heated or when anesthetics or alcohols were added. Ethanol induced the K-to-L uncoiling even at 5³C, much below the denaturation temperature [5]. In contrast, myristate and other luciferin competitors [5] protected the enzyme from thermal denaturation. A similar change in the secondary structure was seen in a simpler polypeptide, poly(L-lysine) [16]. FTIR and the mean residue ellipticity of circular dichroism showed that addition of ethanol increased the L-structure and decreased the K-helix content. During the study, we noticed that the inhibitory potency of fatty acids on ¢re£y luciferase suddenly decreased at the carbon chain length about eight. The inhibitor concentration that decreased the light intensity 50% (IC50 ) of short-chain fatty acids jumped from WM to mM ranges. Short-chain fatty acids decreased the thermal transition temperature. The mode became identical to that of anesthetics and alcohols. The present study compared the e¡ects of butyrate (C4), hexanoate (C6), and octanoate (C8) with those of the longer chain fatty acids on the thermal denaturation temperature and the steadystate luminescence intensity of ¢re£y luciferase. Alcohols also lose the inhibitory potency at carbon chain length about 8^10, which is known as the cuto¡ point. In the alcohol series, however, it is the longer alcohols that lose their potency.
N3I1 3I2 3 ::: 3In 3U ! D where N is the native enzyme, I1 , I2 ... In are the intermediate states, U is the reversibly unfolded state and D is the irreversibly denatured state. Kinetic
2. Method Lyophilized crystalline ¢re£y luciferase from Pho-
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tinus pyralis, sodium salt of D-luciferin, glycylglycine, ATP, and the sodium salts of fatty acids between butyrate (C4) and octadecanoate (C18), were obtained from Sigma (St. Louis, MO). 2.1. Bioluminescence Fire£y luciferase and luciferin were dissolved in 100 mM glycylglycine bu¡er pH 7.8 and placed in a 1.0 ml cuvette. Na2 ATP with MgSO4 solution was rapidly injected into the ¢re£y luciferase solution. The ¢nal concentrations were ¢re£y luciferase 20 Wg ml31 , luciferin 100 WM, ATP 5.0 mM, and MgSO4 10 mM in a total 600 Wl. The fatty acids were added in both solutions. The light output was measured by a Hitachi-Perkin Elmer 139 Spectrophotometer (Norwalk, CT). The photomultiplier output was monitored by a Nicolet 310 Digital Recording Oscilloscope (Madison, WI) and downloaded on £oppy disks. The light output was integrated by software ORIGN (MicroCal, Northampton, MA). 2.2. Thermal transition The total heat absorbed equals the calorimetrically obtained denaturation enthalpy, vHcal . However, irreversibility distorts the DSC thermogram. Therefore, the denaturation temperature was estimated at the temperature where the irreversible denaturation is half completed, T1=2 . Because irreversible denaturation is kinetically controlled, T1=2 varies by the
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scan rate. The thermogram shifts to lower temperatures when the protein concentration is higher or the scan rate is slower [5^7]. We found that the ¢re£y luciferase solution suddenly becomes turbid at the thermal denaturation temperature [6,7]. We compared the turbidity data with those of DSC, obtained by a MicroCal MC2 di¡erential scanning calorimeter (Northampton, MA) at the same scan rate of 1.0³C min31 and the same ¢re£y concentration of 3.0 mg ml31 in 0.5 M glycylglycine bu¡er pH 7.8. The ¢re£y luciferase concentration in the DSC study was higher than in the light intensity study, because the DSC study requires higher protein concentrations to obtain reproducible results [6,7]. When measured by the turbidity, tracings of various scanning rates were parallel to each other [6]. The di¡erential of the turbidity scan was almost the same as the DSC thermogram (Fig. 1). The peak was at 38.1³C with a half-height width of 3.5³C. The scanning rate did not a¡ect the halfheight width of the transition. For a reversible transition, the slowest scan rate has been chosen to maintain a quasi-equilibrium condition during the ¢rstorder transition. However, no matter how slow the scan rate is maintained, or how small the sample size is reduced, one cannot maintain a thermodynamically perfect equilibrium condition represented by zero half-height temperature width. Nevertheless this does not mean that the transition is second order. With irreversible transition, some investigators [11,13] suggested to use a faster scan rate on the ground that the irreversible step occurs at temper-
Table 1 E¡ects of fatty acids on the IC50 , Hill number (nH ), the concentrations that decreased (9 C8) or increased (v C10) phase transition temperature (vT1=2 ) by 1.0³C, and the logarithm of octanol/water partition coe¤cients, log P C4 C6 C8 C10 C12 C14 C16 C18
IC50
naH
vTb1=2
13.6 mM 3.4 mM 2.9 mM 13.2 WM 1.2 WM 0.68 WM 0.67 WM 0.63 WM
3.02 1.04 0.81 0.83 0.89 0.89 0.95 0.95
10.34 mM 3.28 mM 2.46 mM 7.4 WM 2.3 WM 1.2 WM 1.0 WM 0.87 WM
a
log Pc 32.70 31.70 30.70 0.30 1.30 2.31 3.31 4.31
Hill number. Ligand concentrations that changed the transition temperature 1.0³C. Fatty acids with carbon chain lengths shorter than C8 decreased and those longer than C10 increased the T1=2 . c Hansch and Clayton [27]. b
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atures above the reversible transition. It is expected that when the scan rate is faster, there may be less chance of the irreversible change a¡ecting the reversible process. Whatever the rationale, our study showed that the half-height temperature width of the transition did not vary by a scan rate between 2.0 and 0.1³C min31 . The e¡ects of modi¢ers on the denaturation temperature can be meaningfully compared when the scan rates and protein concentrations are the same. The transition temperature, T1=2 , was measured by the turbidity with the solution containing ¢re£y luciferase 0.8 mg ml31 in 100 mM glycylglycine bu¡er pH 7.8. The heating scan rate was 1.0³C min31 . The transmittance was measured at 400 nm in a 1.0 cm light path cuvette by a Perkin Elmer 554 Spectrophotometer (Norwalk, CT) equipped with a programmable digital linear temperature controller and a Peltier heat exchanger. The actual temperature in the sample cuvette was measured by a ¢lament thermistor (YSI, Yellow Springs, OH) inserted into the cuvette and a DigiTech Model 5810 digital thermometer (United Systems, Dayton, OH) with 0.01³C resolution. The light output and the temperature were monitored by the digital recording oscilloscope and downloaded on £oppy disks.
Fig. 1. Comparison of the di¡erential scanning calorimetry thermogram with the temperature scan of spectrophotometry. The ¢re£y luciferase concentration was 3.0 mg ml31 in 0.5 M glycylglycine bu¡er pH 7.8 and the scan rate was 1.0³C min31 . Continuous line: DSC thermogram; broken line: di¡erential of the transmittance at 400 nm.
Fig. 2. IC50 values of fatty acids on the steady-state light intensity of ¢re£y luciferase. Ordinate: the logarithm of the Wmolar concentrations of fatty acids; abscissa: the number of carbon atoms.
3. Results 3.1. Light intensity The steady-state light intensity was estimated from the slope of the linear part of the integral of the light output. The IC50 values decreased with the elongation of the carbon chain: C16: 0.67 WM, C14: 0.68 WM, C12: 11.2 WM, C10: 13.2 WM, C8: 2.9 mM, C6: 3.4 mM, and C4: 13.6 mM (Fig. 2, Table 1). There is a sudden discontinuous change from WM to mM at C10-C8. The IC50 values changed from 13.2 WM of C10 to 2.9 mM of C8, a 220-fold jump in IC50 . In contrast, the change in IC50 from C12 to C10 was only 11-fold. The dose-response curves of the three fatty acids above C8 are shown in Fig. 3. The dose-response curves are not parallel. This is unusual because the dose-response curves of similar compounds such as 1-alkanol series are parallel. The dose-response curves of the fatty acids longer than C10 were parallel [6,7]. By assuming n molecules of the ligand L bind ¢re£y luciferase E with association constant K, the interaction is written K
E nL 3 ELn and
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K
ELn ELn
1
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Fig. 3. Dose-response curves of C8, C6, and C4 on light intensity. Ordinate: the ratio of the steady-state light intensity with and without the inhibitors; abscissa: the logarithm of the molar concentrations of inhibitors. Triangles, C8; squares, C6; circles, C4. Each point is the average of ¢ve determinations and standard errors are inside of the symbols.
ELn KL E n
2
The total enzyme concentration is E0 E ELn
3
Then KLn
ELn E0 3ELn
4
Fig. 5. Dose-dependent e¡ect of C8 on the denaturation temperature. Ordinate: the transmittance of the ¢re£y luciferase solution at 400 nm; abscissa: the temperature of the solution. The scan rate was 1.0³C min31 . The C8 concentrations are from the left: control (thick line), 0.25, 1.0, 4.19, 9.98, and 19.98 mM.
where [E0 ]3[ELn ] is the light intensity in the presence of inhibitor. [ELn ] is the portion where the light intensity is inhibited. We designate the control light intensity without the inhibitor by I1 , and the light intensity with the inhibitor by I2 . Then, [ELn ] = I1 3I2 and [E0 ]3[ELn ] = I2 , and Eq. 4 becomes I 1 3I 2 KLn I2 By taking the logarithm, I1 ln 31 n lnL3ln K I2
Fig. 4. Hill plot. The inhibitor e¡ects are plotted according to Eq. 6 in the text. Ordinate: the logarithm of (I1 /I2 31) where I1 and I2 are the light intensities of ¢re£y luciferase in the absence and presence of the inhibitors, respectively; abscissa : the logarithm of the molar concentrations of the inhibitors. Triangles, C8; squares, C6; circles, C4.
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5
6
At constant temperature and pressure, ln K becomes constant. By plotting the left-hand side to the ligand concentrations, a straight line is obtained. The slope of the straight line indicates the Hill number, nH , of ligand molecules that bind to ¢re£y luciferase. The sigmoidal dose-response curves were linearized according to the above equation and are shown in Fig. 4. The Hill numbers were: C8: 0.812, C6: 1.038, and C4: 3.017. The nH values of the fatty acids longer than C8 were less than one (Table 1). 3.2. Thermal transition temperature The fatty acids with longer carbon chain (v C8)
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Fig. 6. Dose-dependent e¡ect of C6 on the denaturation temperature. The C6 concentrations are from the left: 14.97, 10.14, 5.06, 2.54, 1.27 mM and control (thick line).
increased the thermal denaturation temperature. In contrast, shorter chain fatty acids (9 C6) decreased it. Figs. 5 and 6 show the e¡ects of C8 and C6 on the temperature scan, respectively. The dose-dependent e¡ects of C8, C6, and C4 on the thermal denaturation temperature are shown in Fig. 7. C8 increased T1=2 by 1.0³C at 2.46 mM. C6 and C4 decreased T1=2 by 1.0³C at 3.28 and 10.34 mM, respectively. The fatty acids longer than C10 increased T1=2 . The concentrations that increased T1=2 by 1.0³C were: C10: 7.4 WM, C12: 2.3 WM, C14: 1.2 WM, C16: 1.0 WM, and C18: 0.87 WM (Fig. 8, Table 1).
Fig. 7. Dose-dependent e¡ects of the short-chain fatty acids on the denaturation temperature. Ordinate: change of the denaturation temperature in ³C; abscissa: solution temperature. Triangles, C8; squares, C6; circles, C4. C8 increased, whereas C6 and C4 decreased the denaturation temperature.
Fig. 8. Concentrations of the inhibitor that changed the denaturation temperature T1=2 , by 1.0³C. Ordinate: log concentrations of the inhibitor in Wmolar that changed T1=2 by 1.0³C; abscissa: the number of carbon atoms in the fatty acids. Open circles are the fatty acids hat increased T1=2 . Closed circles are those that decreased T1=2 .
4. Discussion We [5^7] have shown that long-chain fatty acids are strong speci¢c inhibitors of ¢re£y luciferase in competition with luciferin. The initial step of the light emission involves activation of luciferin by ATP to form acyl-AMP between the carboxyl moiety of luciferin and phosphate moiety of AMP. Luciferin is a heterocyclic carboxylate. The initial step of oxidation of fatty acids by acyl-CoA transferases also involves activation of fatty acids by ATP to form acyl-AMP. The homology between the two has been reported [17^19]. It is not surprising that fatty acids compete with luciferin. There is no structural similarity between luciferin and anesthetics. It would be surprising if anesthetics competed with luciferin. To our knowledge, all reports on anesthetic e¡ects on enzymes concluded allosteric interaction. The only exception was by Franks and Lieb [10] who reported that anesthetics compete with luciferin by the use of Lineweaver-Burk plots on the initial £ash intensity in ¢re£y luciferase. Aside from the use of the non-steady-state condition in analyzing the inhibition kinetics, the use of the Lineweaver-Burk plot in multiple-stage reactions requires justi¢cation for the applicability of the linearization procedure. With ¢re£y luciferase, the released free enzyme E and ¢nal
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product oxyluciferin react in the presence of ATP [2,20]. E Oxyluciferin ATP3EWOxylucyferylAMP PPi The multiplicity of the reaction of ¢re£y luciferase invalidates the applicability of the Lineweaver-Burk plot that deals with one substrate and one inhibitor. The above equation shows that oxyluciferin becomes an additional inhibitor. Even if the plot shows apparent competition, the exact binding mode may not be established in this enzyme reaction with two inhibitors and three substrates. The present study showed that there are two modes in the action of fatty acids on ¢re£y luciferase. The action mode changed at the carbon chain length about C8. The IC50 increased from 13.2 WM of C10 to 2.9 mM of C8, a 220-fold jump. The change from C12 to C10 was 11-fold. A more dramatic change was observed with the transition temperature, where those longer than C10 increased the transition temperature, whereas those shorter than C8 decreased the transition temperature. The concentration that changed the transition temperature 1.0³C (negative or positive) increased from 7.4 WM of C10 to 2.46 mM of C8, a 332-fold jump. The change from C12 to C10 was 3.2-fold. The dose-response curves showed a large change as shown in Fig. 3. The Hill numbers changed from less than one at C8 to more than one at C6. The Hill number assumes a highly cooperative interaction between enzyme E and n molecules of ligand L to yield only one ELn molecule. The intermediates, EL1 , EL2 ... ELn31 are ignored. Therefore nH s 1 means that at least two or more ligand molecules are cooperatively bound to the enzyme. Conversely, nH 91 means noncooperative limited binding. The real binding number must be estimated by the Scatchard plot. It is possible that the actual binding numbers of the ligand with nH s 1 are much more than the Hill number. We reported by 19 F-NMR [21] that the maximum binding number, Bmax , of halothane to bovine serum albumin (BSA) was 34, and by di¡erential titration calorimetry [22] that the Bmax of chloroform, bound to BSA, was 13.2. Both values were obtained by the Scatchard plot. The present result shows that the binding number suddenly increased when the carbon chain length becomes shorter than
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C8. The binding mode changed from a non-cooperative limited number of binding sites to a highly cooperative multiple binding sites. Koshland [23,24] proposed the induced-¢t model where the ligands that bind to the substrate binding sites induce the protein structure to a high-energy native state. Eyring [25,26] proposed the rate process model where anesthetics and alcohols bind non-speci¢cally to proteins and reversibly unfold the protein into a less active state by releasing the surface-bound water molecules. Fatty acids longer than C10 are speci¢c binders to the high-energy native state of ¢re£y luciferase. Short-chain fatty acids are non-speci¢c binders to the reversibly unfolded enzyme. The sign of the logarithm of the oil/water partition coe¤cients (log P) of the short-chain fatty acids changes from positive (C10) to negative (C8) at the identical position where the change from the speci¢c to non-speci¢c interaction occurred. The negative sign signi¢es that the short-chain fatty acids distribute more to the aqueous phase than to the organic phase (oil/water 6 1). Due to the strong hydrophobicity of the luciferin binding site [2], shorter fatty acids are apparently excluded from the luciferin binding site. Acknowledgements Supported by DVA Medical Research Funds.
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[18] P.C. Babbitt, G.L. Kenyon, B.M. Martin, H. Charest, M. Slyvestre, J.D. Scholten, K.H. Chang, P.H. Liang, D. Dunaway-Mariano, Biochemistry 31 (1992) 5594^5604. [19] L. Ye, L.M. Buck, H.L. Schae¡er, F.R. Leach, Biochim. Biophys. Acta 1339 (1997) 39^52. [20] R. Lee, W.D. McElroy, Biochemistry 8 (1969) 130^136. [21] T. Yoshida, M. Tanaka, Y. Mori, I. Ueda, Biochim. Biophys. Acta 1334 (1997) 117^122. [22] I. Ueda, M. Yamanaka, Biophys. J. 72 (1997) 1812^1817. [23] D.E. Koshland Jr., Proc. Natl. Acad. Sci. USA 44 (1958) 98^104. [24] D.E. Koshland Jr., Science 142 (1963) 1533^1541. [25] H. Eyring, J.L. Magee, J. Cell Comp. Physiol. 20 (1942) 169^177. [26] H. Eyring, Science 154 (1966) 1609^1613. [27] C. Hansch, J.M. Clayton, J. Pharm. Sci. 62 (1973) 1^21.
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