- Email: [email protected]
Kinetics of irreversible inhibition of yeast alcohol dehydrogenase during modification by 4,4′-dithiodipyridine
International Journal of Biological Macromolecules 20 (1997) 307 – 313
Kinetics of irreversible inhibition of yeast alcohol dehydrogenase during modification by 4,4%-dithiodipyridine Si-Yang Zheng 1, Dong Xu, Hong-Rui Wang 2, Jian Li, Hai-Meng Zhou * Department of Biological Science and Biotechnology, Tsinghua Uni6ersity, Beijing 100084, China Received 13 November 1996; accepted 21 April 1997
Abstract The course of inactivation of yeast alcohol dehydrogenase (YADH) using 4,4%-dithiodipyridine (DSDP) has been studied in this paper. The results show that the reaction mechanism between DSDP and YADH is a competitive, complexing inhibition. The microscopic constants for the inactivation of the free enzyme and the enzyme-substrate complex were determined. The presence of the substrate NAD + offers strong protection for this enzyme against inactivation by DSDP. The above results suggest that two Cys residues are essential for activity and are situated at the active site. These essential Cys residues should be Cys-46 and Cys-174 which are ligands to the catalytic zinc ion. Another Cys residue, which can be modified by DSDP, is non-essential for activity of the enzyme. © 1997 Elsevier Science B.V. Keywords: Alcohol dehydrogenase; Irreversible inhibition; Inactivation; 4,4%-Dithiodipyridine
1. Introduction Yeast alcohol dehydrogenase (alcohol: NAD + oxidoreductase, EC 1.1.1.1) is a tetrameric enAbbre6iations: DSDP, 4,4%-dithiodipyridine; EtOH, ethanol; NAD + , nicotinamide adenine dinucleotide; OPTA, o-phthalaldehyde; YADH, yeast alcohol dehydrogenase. * Corresponding author. Fax: + 86 10 62568182. 1 Present address: Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA. 2 Present address: Division of Biology, 147–75, California Institute of Technology, Pasadena, CA 91125, USA.
zyme [1,2]. The active site at each subunit contains a zinc ion which is absolutely necessary for enzyme activity [3]. The second zinc ion present on each enzyme subunit plays a prominent conformational role, probably by stabilizing the tertiary structure of the enzyme [4]. The presence of the conformational zinc in YADH helps to fold the molecule and maintain the native conformation of the enzyme [5,6]. It is well known that the Cys-46 and Cys-174 of YADH located at the active site are essential for enzyme activity, and are ligands bound to the catalytic zinc ion [3]. The
0141-8130/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 4 1 - 8 1 3 0 ( 9 7 ) 0 0 0 3 0 - 5
308
S.-Y. Zheng et al. / International Journal of Biological Macromolecules 20 (1997) 307–313
Table 1 Kinetic constants for the inhibtion of yeast alcohol dehydrogenase by 4,4%-dithiodipyridine V (mM/min)
KI (mM)
K %I (mM)
ki (s−1)
k %i (s−1)
(k+0 ¦ , k4+k+0 § , k3)/(k3+k4) (s min−1)
57.4
2.0×10−2
7.8×10−2
1.9×10−3
0
0
inactivation of YADH was studied by following the generation of NADH in the presence of o-phthalaldehyde (OPTA) [7]. OPTA can form a fluorescence group in a thiol-containing protein [8]. The results in the present paper show that the inactivation of YADH by DSDP is slow and irreversible. The inhibition mechanism of the DSDP reaction is a complexing, competitive, and irreversible inhibition. The results also show the marked protective effect of NAD + on the inactivation of YADH by DSDP. Therefore, it is suggested that the modification may occur at the active site of the enzyme.
tive, the number of modified SH group was calculated directly from the molar ratio DSDP/enzyme in the reaction solution after the modification reaction was finished. The progress-of-substrate-reaction method [11] was used for the study of the inactivation kinetics of YADH by DSDP. In this method, 20 ml of 0.1 mM YADH was added to 1.0 ml of an assay system containing different concentrations of DSDP, and the progress curve for the substrate reaction was analyzed [11,12] to obtain the rate constants (Table 1). All measurements were carried out at 25°C.
2. Material and methods
3. Results
Yeast alcohol dehydrogenase was obtained from Sigma and used without further purification. The sodium salt of NAD + was from Boehringer Mannham GmbH. The 4,4%-dithiodipyridine was also obtained from Sigma. All other reagents were local products of analytical grade. Enzyme concentration was determined by measuring the absorbency at 280 nm using the absorption coefficient A 1% 1 cm =12.6 [9]. The enzyme activity was assayed with a Kontron Uvikon 860 spectrophotometer using the method described by Bille and Remade [10]. The enzyme was studied with DSDP in 0.1 M phosphate buffer (pH =7.2) at 25°C. The modification reaction of the enzyme was followed using the spectral changes between 300 – 400 nm. After the reaction was finished, the remaining enzyme activity was measured by adding 10 ml of the reaction mixture to 1 ml of an assay system. The assay system contained 0.1 M phosphate buffer (pH 7.5), 0.015 M sodium pyrophosphate, 0.15 M EtOH and 0.75 mM NAD + . Since the reaction of DSDP with SH groups in a protein is quantita-
3.1. Modification of yeast alcohol dehydrogenase by DSDP Spectral changes occur when DSDP binds to the sulfhydryl group of a protein [13]. DSDP binding to YADH produces a spectrum with a peak at 324 nm. Thus the kinetic course of DSDP binding to YADH was followed using the absorbance change at 324 nm (Fig. 1). It can be seen that P(A324) increases with increasing time until a constant final value [P] is approached. A semilogarithmic plot (show in the insert) gives a straight line, indicating that the reaction is monophasic. This result suggests that all reactive thiol groups in the enzyme molecule react with DSDP at the same rate.
3.2. The number of reacti6e and essential thiol groups in YADH during DSDP modification Yeast alcohol dehydrogenase is a tetrameric enzyme composed of identical subunits, each of which contains 9 cysteine residues [14]. The absorption spectra of the modified enzyme during
S.-Y. Zheng et al. / International Journal of Biological Macromolecules 20 (1997) 307–313
309
Fig. 1. Course of the modification reaction of YADH using DSDP. The enzyme was modified in 25 mM PBS buffer (pH= 7.4) containing 2.17 mM DSDP at 25°C. The final concentration of the enzyme was 2.1 nM. The absorption change at 324 nm was followed after adding the modifier. The inset shows a semilogarithmic plot.
treatment with different amounts of DSDP are shown in Fig. 2. An increase in the amount of DSDP leads to an increase in the absorbance peak at 324 nm until a constant maximum value is
approached. The results show that each subunit of the enzyme contains three reactive thiol groups. The native enzyme was treated with different amounts of DSDP, and then the remaining enzyme activity was determined. Fig. 3 shows that relationship between the fractional activity remaining (a) and the extent of modification of the reactive thiol groups (m). It can be seen from Fig. 3 that when i= 2, the plot of a 1/i against m gives a straight line, indicating that only two of the three reactive groups in each subunit are essential for activity of the enzyme [11]
3.3. Kinetics of inacti6ation of YADH by DSDP
Fig. 2. Absorbance spectra of enzyme modified using different amounts of DSDP. Experimental condition as for Fig. 1 except for the DSDP concentrations. The spectra were determined after modification for 24 h. Final enzyme concentration was 2.8 nM. For curves 1–8, the molar ratio of DSDP/subunit of the enzyme was 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 4.0, respectively.
The time courses of the substrate reaction in the presence of different concentrations of DSDP are shown in Fig. 4a. It could be seen that [P] approaches constant final values, [P] , which decrease with increasing concentrations of DSDP. The concentration of the product formed at time t can be expressed [11,16] as: ln([P] − [P])= ln[P] − A[Y]t
310
S.-Y. Zheng et al. / International Journal of Biological Macromolecules 20 (1997) 307–313
4b. The apparent rate constant, A, can be calculated from the slopes of the straight lines. The results show that the inactivation is a monophasic pseudo-first-order reaction. It has been pointed out that plots of 1/A against [Y] can be used to differentiate complexing from noncomplexing inhibition reactions [11,12]. A plot of 1/A against [DSDP] shows that the apparent rate constant, A, is dependent on [DSDP], as shown in Fig. 4c, indicting that DSDP is a complexing irreversible inhibitor. In addition, a plot of the final product concentration, [P] , against 1/[DSDP] gives a straight line through the origin (figure not shown), indicating that DSDP is a competitive irreversible inhibitor [11].
3.4. Determination of microscopic rate constants Fig. 3. The fractional remaining activity (a) to the 1/i power plotted as a function of the extent of modification of the reactive thiol group (m) in each subunit of the enzyme according to Tsou [12] when i = 1 ( ), i= 2(), i= 3(). Final concentration of the enzyme was 2.0 mM.
where [P] is the product concentration at time infinity. A is the apparent rate constant of inactivation, and [Y] is the concentration of the inhibitor. YADH reaction involving two substrates with the order Bi–Bi mechanism are shown below:
dissociation conThe rate constants (ki, k %), i stants (KI, K %) and apparent rate constant I ¦ 0, k4 + k + § 0, k3)/(k3 + k4) can be obtained from (k + measurement of the substrate reaction in the presence of DSDP at different substrate concentrations. Km, Vmax and KiR (or KiNAD + , the dissociation constant of E-NAD + ) are known quantities [8] which can be measured in the absence of DSDP. Plots of 1/[P] against 1/[EtOH] are shown in Fig. 5a. The slope, s, and the intercept, i, obtained from Fig. 5a are then plotted against 1/[NAD + ] in Fig. 5b. The initial velocity of the modified enzyme reaction, 6* can be obtained from 6*= A[Y][P] . Plots of 1/6* against 1/[EtOH] give a series of straight lines as shown in Fig. 6a. The slope and intercept obtained from Fig. 6a are then plotted against 1/[NAD + ] in Fig. the 6b. The microscopic rate constants (ki, k %), i dissociation constants (KI, K %) and the value of V I was then derived from the secondary plots given in Fig. 5b and Fig. 6b. The results are summarized in Table 1.
4. Discussion
Plots of ln([P] −[P]) against time t give a series of straight lines at different concentrations of DSDP with slopes of − A[Y] as shown in Fig.
Yeast alcohol dehydrogenase is a tetrameric enzyme containing zinc ions. The presence of two zinc ions per subunit has been confirmed. One is found in the catalytic site of the enzyme [4].
S.-Y. Zheng et al. / International Journal of Biological Macromolecules 20 (1997) 307–313
311
Fig. 4. Substrate reaction of YADH in the presence of different concentrations of DSDP. (a) Time courses of substrate reaction of YADH in the presence of DSDP. The reaction mixture contained 0.1 M phosphate buffer, pH =7.5, 0.015 M sodium pyrophosphate, 0.1 M EtOH, 0.5 mM NAD + and 2 nM YADH. For curves 1 – 6, the concentrations of DSDP were 0, 30, 42, 60, 90 and 120 mM. NADH generation was monitored by the absorbance at 340 nm and 25°C. (b) Semilogarithmic plot of the slope, s, of lines 2 – 6 according to equation (1). (c) A plot of 1/A against concentration of DSDP. The valves of A were calculated from Fig. 4b. (d) A plot of P (A340) against 1/[DSDP].
Cys-46 and Cys-174 of YADH, located at the active site, are ligands to the catalytic zinc ion and are essential for activity of the enzyme [3]. In the present investigation, Tsou’s method [11] is applied to study the kinetics of the course of inactivation of YADH which has two substrates with
the ordered Bi–Bi reaction mechanism. There are two types of irreversible inhibitions, complexing and non-complexing types. The complexing inhibitor [Y] can form a noncovalent reversible complex with enzyme (E), forming EY, prior to an irreversible modification step leading to EY%.
312
S.-Y. Zheng et al. / International Journal of Biological Macromolecules 20 (1997) 307–313
Fig. 5. Determination of the microscopic rate constants of the inactivation of YADH. (a) Effect of EtOH concentration on [P] during inactivation by DSDP. Experimental conditions were as for Fig. 4 except that the concentration of DSDP and YADH were 112.5 mM and 2.1 nm, respectively, and the concentration of EtOH and NAD + were as indicated. P represents the product. 1/[P] was plotted against 1/[EtOH] with [NAD + ] of 0.75, 1.00, 1.125, 1.50, and 1.875 mM for lines 1 – 5, respectively. (b) Secondary plot of the ordinate intercept, i () (right-hand scale) and the slopes, s (), (left-hand scale) against 1/[NAD + ].
The difference between apparent rate constants for complexing and noncomplexing inhibitors is that the expression for the apparent rate constant A contains the term [Y] for complexing inhibitors, whereas it is independent of [Y] for noncomplex-
ing inhibitors [15,16]. This provides the basis for the experimental differentiation of this two types of inhibitions. It was previously reported that for inactivation of YADH by OPTA, a plot of 1/A against [Y] gives a line parallel to the abscissa,
S.-Y. Zheng et al. / International Journal of Biological Macromolecules 20 (1997) 307–313
313
Fig. 6. Determination of the microscopic rate constants of the inactivation of YADH. (a) Effect of EtOH concentration on 1/6* during inactivation by DSDP. Experimental conditions were as for Fig. 5. 1/6* was plotted against 1/[EtOH] with [NAD + ] of 0.75, 1.00, 1.125 and 1.50 mM for lines 1–4, respectively. b) Secondary plots of the ordinate intercepts, i () (right-hand scale) and slopes, s ( ) (left-hand scale) against 1/[NAD + ].
indicating that A is independent of [Y] [16] and that the inactivation of YADH by OPTA is a noncomplexing irreversible inhibition. The results of the present investigation show that inactivation of YADH by DSDP is a complexing type inhibition. However, both OPTA and DSDP are competitive inhibitors. The results also show the marked protective effect of NAD + on the inactivation of YADH by these two inhibitors. Therefore, it is suggested that these two inhibitors modify the active site of YADH.
Acknowledgements The present investigation was partly supported by the Pandeng Project of the China Commission for Science and Technology.
References [1] Veillon C, Sytkowski AJ. Biochem Biophys Res Commun
1975;67:1494 – 500. [2] Klinman JP, Welsh K. Biochem Biophys Res Commun 1976;70:878 – 84. [3] Coleman PL, Weiner H. Biochemistry 1973;12:3466 – 72. [4] Magonet E, Hayen P, Delforge D, Delaive E. Biochem J 1992;287:361 – 5. [5] Le WP, Yan SX, Zhang YX, Zhou HM. J Biochem 1996;119:674 – 9. [6] Le WP, Yan SX, Li S, Zhong HN, Zhou HM. Int J Peptide Protein Res 1996;47:484 – 90. [7] Le WP, Yan SX, Huang MQ, Zhang YX, Zhou HM. Enzyme Protein 1995;48:183 – 90. [8] Palczewski K, Hargrave P, Kochman M. Eur J Biochem 1983;137:429 – 35. [9] Hayes JI Jr., Velik SF. J Biol Chem 1954;207:225 – 44. [10] Bille V, Remacle J. Eur J Biochem 1986;160:343 – 8. [11] Tsou CL. Adv Enzymol Relat Areas Mol Biol 1988;61:381 – 436. [12] Tian WX, Tsou CL. Biochemistry 1982;21:1028 – 32. [13] Grassett DR, Murry JP Jr. Arch Biochem Biophys 1967;119:41 – 9. [14] Eklund H, Braarden CI. Zinc Enzyme, New York: John Wiley, 1983:124 – 152 [15] Wang ZX, Tsou CL. J Theor Biol 1987;127:253 – 70. [16] Wang ZX, Preiss B, Tsou CL. Biochemistry 1988;27:5095 – 100.
Kinetics of irreversible inhibition of yeast alcohol dehydrogenase during modification by 4,4%-dithiodipyridine Si-Yang Zheng 1, Dong Xu, Hong-Rui Wang 2, Jian Li, Hai-Meng Zhou * Department of Biological Science and Biotechnology, Tsinghua Uni6ersity, Beijing 100084, China Received 13 November 1996; accepted 21 April 1997
Abstract The course of inactivation of yeast alcohol dehydrogenase (YADH) using 4,4%-dithiodipyridine (DSDP) has been studied in this paper. The results show that the reaction mechanism between DSDP and YADH is a competitive, complexing inhibition. The microscopic constants for the inactivation of the free enzyme and the enzyme-substrate complex were determined. The presence of the substrate NAD + offers strong protection for this enzyme against inactivation by DSDP. The above results suggest that two Cys residues are essential for activity and are situated at the active site. These essential Cys residues should be Cys-46 and Cys-174 which are ligands to the catalytic zinc ion. Another Cys residue, which can be modified by DSDP, is non-essential for activity of the enzyme. © 1997 Elsevier Science B.V. Keywords: Alcohol dehydrogenase; Irreversible inhibition; Inactivation; 4,4%-Dithiodipyridine
1. Introduction Yeast alcohol dehydrogenase (alcohol: NAD + oxidoreductase, EC 1.1.1.1) is a tetrameric enAbbre6iations: DSDP, 4,4%-dithiodipyridine; EtOH, ethanol; NAD + , nicotinamide adenine dinucleotide; OPTA, o-phthalaldehyde; YADH, yeast alcohol dehydrogenase. * Corresponding author. Fax: + 86 10 62568182. 1 Present address: Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA. 2 Present address: Division of Biology, 147–75, California Institute of Technology, Pasadena, CA 91125, USA.
zyme [1,2]. The active site at each subunit contains a zinc ion which is absolutely necessary for enzyme activity [3]. The second zinc ion present on each enzyme subunit plays a prominent conformational role, probably by stabilizing the tertiary structure of the enzyme [4]. The presence of the conformational zinc in YADH helps to fold the molecule and maintain the native conformation of the enzyme [5,6]. It is well known that the Cys-46 and Cys-174 of YADH located at the active site are essential for enzyme activity, and are ligands bound to the catalytic zinc ion [3]. The
0141-8130/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 4 1 - 8 1 3 0 ( 9 7 ) 0 0 0 3 0 - 5
308
S.-Y. Zheng et al. / International Journal of Biological Macromolecules 20 (1997) 307–313
Table 1 Kinetic constants for the inhibtion of yeast alcohol dehydrogenase by 4,4%-dithiodipyridine V (mM/min)
KI (mM)
K %I (mM)
ki (s−1)
k %i (s−1)
(k+0 ¦ , k4+k+0 § , k3)/(k3+k4) (s min−1)
57.4
2.0×10−2
7.8×10−2
1.9×10−3
0
0
inactivation of YADH was studied by following the generation of NADH in the presence of o-phthalaldehyde (OPTA) [7]. OPTA can form a fluorescence group in a thiol-containing protein [8]. The results in the present paper show that the inactivation of YADH by DSDP is slow and irreversible. The inhibition mechanism of the DSDP reaction is a complexing, competitive, and irreversible inhibition. The results also show the marked protective effect of NAD + on the inactivation of YADH by DSDP. Therefore, it is suggested that the modification may occur at the active site of the enzyme.
tive, the number of modified SH group was calculated directly from the molar ratio DSDP/enzyme in the reaction solution after the modification reaction was finished. The progress-of-substrate-reaction method [11] was used for the study of the inactivation kinetics of YADH by DSDP. In this method, 20 ml of 0.1 mM YADH was added to 1.0 ml of an assay system containing different concentrations of DSDP, and the progress curve for the substrate reaction was analyzed [11,12] to obtain the rate constants (Table 1). All measurements were carried out at 25°C.
2. Material and methods
3. Results
Yeast alcohol dehydrogenase was obtained from Sigma and used without further purification. The sodium salt of NAD + was from Boehringer Mannham GmbH. The 4,4%-dithiodipyridine was also obtained from Sigma. All other reagents were local products of analytical grade. Enzyme concentration was determined by measuring the absorbency at 280 nm using the absorption coefficient A 1% 1 cm =12.6 [9]. The enzyme activity was assayed with a Kontron Uvikon 860 spectrophotometer using the method described by Bille and Remade [10]. The enzyme was studied with DSDP in 0.1 M phosphate buffer (pH =7.2) at 25°C. The modification reaction of the enzyme was followed using the spectral changes between 300 – 400 nm. After the reaction was finished, the remaining enzyme activity was measured by adding 10 ml of the reaction mixture to 1 ml of an assay system. The assay system contained 0.1 M phosphate buffer (pH 7.5), 0.015 M sodium pyrophosphate, 0.15 M EtOH and 0.75 mM NAD + . Since the reaction of DSDP with SH groups in a protein is quantita-
3.1. Modification of yeast alcohol dehydrogenase by DSDP Spectral changes occur when DSDP binds to the sulfhydryl group of a protein [13]. DSDP binding to YADH produces a spectrum with a peak at 324 nm. Thus the kinetic course of DSDP binding to YADH was followed using the absorbance change at 324 nm (Fig. 1). It can be seen that P(A324) increases with increasing time until a constant final value [P] is approached. A semilogarithmic plot (show in the insert) gives a straight line, indicating that the reaction is monophasic. This result suggests that all reactive thiol groups in the enzyme molecule react with DSDP at the same rate.
3.2. The number of reacti6e and essential thiol groups in YADH during DSDP modification Yeast alcohol dehydrogenase is a tetrameric enzyme composed of identical subunits, each of which contains 9 cysteine residues [14]. The absorption spectra of the modified enzyme during
S.-Y. Zheng et al. / International Journal of Biological Macromolecules 20 (1997) 307–313
309
Fig. 1. Course of the modification reaction of YADH using DSDP. The enzyme was modified in 25 mM PBS buffer (pH= 7.4) containing 2.17 mM DSDP at 25°C. The final concentration of the enzyme was 2.1 nM. The absorption change at 324 nm was followed after adding the modifier. The inset shows a semilogarithmic plot.
treatment with different amounts of DSDP are shown in Fig. 2. An increase in the amount of DSDP leads to an increase in the absorbance peak at 324 nm until a constant maximum value is
approached. The results show that each subunit of the enzyme contains three reactive thiol groups. The native enzyme was treated with different amounts of DSDP, and then the remaining enzyme activity was determined. Fig. 3 shows that relationship between the fractional activity remaining (a) and the extent of modification of the reactive thiol groups (m). It can be seen from Fig. 3 that when i= 2, the plot of a 1/i against m gives a straight line, indicating that only two of the three reactive groups in each subunit are essential for activity of the enzyme [11]
3.3. Kinetics of inacti6ation of YADH by DSDP
Fig. 2. Absorbance spectra of enzyme modified using different amounts of DSDP. Experimental condition as for Fig. 1 except for the DSDP concentrations. The spectra were determined after modification for 24 h. Final enzyme concentration was 2.8 nM. For curves 1–8, the molar ratio of DSDP/subunit of the enzyme was 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 4.0, respectively.
The time courses of the substrate reaction in the presence of different concentrations of DSDP are shown in Fig. 4a. It could be seen that [P] approaches constant final values, [P] , which decrease with increasing concentrations of DSDP. The concentration of the product formed at time t can be expressed [11,16] as: ln([P] − [P])= ln[P] − A[Y]t
310
S.-Y. Zheng et al. / International Journal of Biological Macromolecules 20 (1997) 307–313
4b. The apparent rate constant, A, can be calculated from the slopes of the straight lines. The results show that the inactivation is a monophasic pseudo-first-order reaction. It has been pointed out that plots of 1/A against [Y] can be used to differentiate complexing from noncomplexing inhibition reactions [11,12]. A plot of 1/A against [DSDP] shows that the apparent rate constant, A, is dependent on [DSDP], as shown in Fig. 4c, indicting that DSDP is a complexing irreversible inhibitor. In addition, a plot of the final product concentration, [P] , against 1/[DSDP] gives a straight line through the origin (figure not shown), indicating that DSDP is a competitive irreversible inhibitor [11].
3.4. Determination of microscopic rate constants Fig. 3. The fractional remaining activity (a) to the 1/i power plotted as a function of the extent of modification of the reactive thiol group (m) in each subunit of the enzyme according to Tsou [12] when i = 1 ( ), i= 2(), i= 3(). Final concentration of the enzyme was 2.0 mM.
where [P] is the product concentration at time infinity. A is the apparent rate constant of inactivation, and [Y] is the concentration of the inhibitor. YADH reaction involving two substrates with the order Bi–Bi mechanism are shown below:
dissociation conThe rate constants (ki, k %), i stants (KI, K %) and apparent rate constant I ¦ 0, k4 + k + § 0, k3)/(k3 + k4) can be obtained from (k + measurement of the substrate reaction in the presence of DSDP at different substrate concentrations. Km, Vmax and KiR (or KiNAD + , the dissociation constant of E-NAD + ) are known quantities [8] which can be measured in the absence of DSDP. Plots of 1/[P] against 1/[EtOH] are shown in Fig. 5a. The slope, s, and the intercept, i, obtained from Fig. 5a are then plotted against 1/[NAD + ] in Fig. 5b. The initial velocity of the modified enzyme reaction, 6* can be obtained from 6*= A[Y][P] . Plots of 1/6* against 1/[EtOH] give a series of straight lines as shown in Fig. 6a. The slope and intercept obtained from Fig. 6a are then plotted against 1/[NAD + ] in Fig. the 6b. The microscopic rate constants (ki, k %), i dissociation constants (KI, K %) and the value of V I was then derived from the secondary plots given in Fig. 5b and Fig. 6b. The results are summarized in Table 1.
4. Discussion
Plots of ln([P] −[P]) against time t give a series of straight lines at different concentrations of DSDP with slopes of − A[Y] as shown in Fig.
Yeast alcohol dehydrogenase is a tetrameric enzyme containing zinc ions. The presence of two zinc ions per subunit has been confirmed. One is found in the catalytic site of the enzyme [4].
S.-Y. Zheng et al. / International Journal of Biological Macromolecules 20 (1997) 307–313
311
Fig. 4. Substrate reaction of YADH in the presence of different concentrations of DSDP. (a) Time courses of substrate reaction of YADH in the presence of DSDP. The reaction mixture contained 0.1 M phosphate buffer, pH =7.5, 0.015 M sodium pyrophosphate, 0.1 M EtOH, 0.5 mM NAD + and 2 nM YADH. For curves 1 – 6, the concentrations of DSDP were 0, 30, 42, 60, 90 and 120 mM. NADH generation was monitored by the absorbance at 340 nm and 25°C. (b) Semilogarithmic plot of the slope, s, of lines 2 – 6 according to equation (1). (c) A plot of 1/A against concentration of DSDP. The valves of A were calculated from Fig. 4b. (d) A plot of P (A340) against 1/[DSDP].
Cys-46 and Cys-174 of YADH, located at the active site, are ligands to the catalytic zinc ion and are essential for activity of the enzyme [3]. In the present investigation, Tsou’s method [11] is applied to study the kinetics of the course of inactivation of YADH which has two substrates with
the ordered Bi–Bi reaction mechanism. There are two types of irreversible inhibitions, complexing and non-complexing types. The complexing inhibitor [Y] can form a noncovalent reversible complex with enzyme (E), forming EY, prior to an irreversible modification step leading to EY%.
312
S.-Y. Zheng et al. / International Journal of Biological Macromolecules 20 (1997) 307–313
Fig. 5. Determination of the microscopic rate constants of the inactivation of YADH. (a) Effect of EtOH concentration on [P] during inactivation by DSDP. Experimental conditions were as for Fig. 4 except that the concentration of DSDP and YADH were 112.5 mM and 2.1 nm, respectively, and the concentration of EtOH and NAD + were as indicated. P represents the product. 1/[P] was plotted against 1/[EtOH] with [NAD + ] of 0.75, 1.00, 1.125, 1.50, and 1.875 mM for lines 1 – 5, respectively. (b) Secondary plot of the ordinate intercept, i () (right-hand scale) and the slopes, s (), (left-hand scale) against 1/[NAD + ].
The difference between apparent rate constants for complexing and noncomplexing inhibitors is that the expression for the apparent rate constant A contains the term [Y] for complexing inhibitors, whereas it is independent of [Y] for noncomplex-
ing inhibitors [15,16]. This provides the basis for the experimental differentiation of this two types of inhibitions. It was previously reported that for inactivation of YADH by OPTA, a plot of 1/A against [Y] gives a line parallel to the abscissa,
S.-Y. Zheng et al. / International Journal of Biological Macromolecules 20 (1997) 307–313
313
Fig. 6. Determination of the microscopic rate constants of the inactivation of YADH. (a) Effect of EtOH concentration on 1/6* during inactivation by DSDP. Experimental conditions were as for Fig. 5. 1/6* was plotted against 1/[EtOH] with [NAD + ] of 0.75, 1.00, 1.125 and 1.50 mM for lines 1–4, respectively. b) Secondary plots of the ordinate intercepts, i () (right-hand scale) and slopes, s ( ) (left-hand scale) against 1/[NAD + ].
indicating that A is independent of [Y] [16] and that the inactivation of YADH by OPTA is a noncomplexing irreversible inhibition. The results of the present investigation show that inactivation of YADH by DSDP is a complexing type inhibition. However, both OPTA and DSDP are competitive inhibitors. The results also show the marked protective effect of NAD + on the inactivation of YADH by these two inhibitors. Therefore, it is suggested that these two inhibitors modify the active site of YADH.
Acknowledgements The present investigation was partly supported by the Pandeng Project of the China Commission for Science and Technology.
References [1] Veillon C, Sytkowski AJ. Biochem Biophys Res Commun
1975;67:1494 – 500. [2] Klinman JP, Welsh K. Biochem Biophys Res Commun 1976;70:878 – 84. [3] Coleman PL, Weiner H. Biochemistry 1973;12:3466 – 72. [4] Magonet E, Hayen P, Delforge D, Delaive E. Biochem J 1992;287:361 – 5. [5] Le WP, Yan SX, Zhang YX, Zhou HM. J Biochem 1996;119:674 – 9. [6] Le WP, Yan SX, Li S, Zhong HN, Zhou HM. Int J Peptide Protein Res 1996;47:484 – 90. [7] Le WP, Yan SX, Huang MQ, Zhang YX, Zhou HM. Enzyme Protein 1995;48:183 – 90. [8] Palczewski K, Hargrave P, Kochman M. Eur J Biochem 1983;137:429 – 35. [9] Hayes JI Jr., Velik SF. J Biol Chem 1954;207:225 – 44. [10] Bille V, Remacle J. Eur J Biochem 1986;160:343 – 8. [11] Tsou CL. Adv Enzymol Relat Areas Mol Biol 1988;61:381 – 436. [12] Tian WX, Tsou CL. Biochemistry 1982;21:1028 – 32. [13] Grassett DR, Murry JP Jr. Arch Biochem Biophys 1967;119:41 – 9. [14] Eklund H, Braarden CI. Zinc Enzyme, New York: John Wiley, 1983:124 – 152 [15] Wang ZX, Tsou CL. J Theor Biol 1987;127:253 – 70. [16] Wang ZX, Preiss B, Tsou CL. Biochemistry 1988;27:5095 – 100.