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1 Vol. 43, No. 2, October 1997 BIOCHEMISTRY end MOLECULAR BIOLOGY INTERNATIONAL Pages NOVEL SUBSTRATES OF YEAST ALCOHOL DEHYDROGENASE - 3, 4-DIMETHYLAMINO-CINNAMALDEHYDE AND CHLOROACETALDEHYDE V. Leskovac 1, S. Trivial 2, j. Zeremski 3 B. Stan~i~: 4 and B.M. Anderson s I Faculty of Technology Novi Sad, Bulevar Cara Lazara 1, Novi Sad, z Faculty of Science Novi Sad, 3 Faculty of Technology Beograd, 4 Faculty of Agriculture Novi Sad, Yugoslavia, and ~ Virginia Polytechnic Institute and State University, Virginia, USA Received May 15, 1997 Received after revision July 23, 1997 SUMMARY: 4.Dimethylamino-trans-cinnamaldehyde and chloroacetaldehyde are novel substrates of yeast alcohol dehydrogenase (EC ). In the present work, we have reported the steady-state kinetic constants for both substrates, and their chemical reactions with the enzyme protein itself. Both substrates are potentially useful for biotechnology, chemoenzyme syntheses and analytical biochemistry. INTRODUCTION Yeast alcohol dehydrogenase (EC , constitutive, cytoplasmic) has a much narrower substrate specificity compared with equine liver enzyme [1]. Yeast en-, zyme catalyzes efficiently reduction of aliphatic aldehydes with NADH, but any branching in the side chain of substrates diminishes the rate of enzymatic reactions [2-4]; aromatic aldehydes are poor substrates of yeast enzyme [5]. In the present work, we have determined the steady-state kinetic properties of two novel substrates of yeast enzyme, 4-dimethylamino-trans-cinnamaldehyde and chloroacetaldehyde. Since both compounds are chemically reactive, we have also investigated their chemical reactions with an enzyme protein itself. Abbreviations: YADH, yeast alcohol dehydrogenase; DACA, 4-dknethylamino-trans-cinnamaldehyde; NDIvlA, p..nitroso-n,/v-dimethylaniline; CE, 2-chloroethanol; CAA, chloroacetaldehyde /97/ /0 Copyright by Academic Press Australia. All rig/ts of reproduction in any form reserved.
2 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL MATERIALS AND METHODS Material.s. Yeast alcohol dehydrogenase (iyophilized) was obtained from Boehringet. Specific activity of enzyme with ethanol was 300 U/mg of enzyme protein, estimated at ph 9, according to Bergmeyer [6]. The concentration of enzyme protein in solution was determined according to Hayes and Velick [7], and the concentration of enzyme active s'ites by the 11uorescent method of Leskovac eta/. [8]. NAD and NADH were purchased from Sigma; chloroacetaldehyde (50 wt.% solution in water), 2-chloroethanol (99%), 4-dimethylamino-trans-cinnamaidehyde (98%) and Tds base (99,9+%) were purchased from Aldrich, and used without further purification. All other chemicals were of the highest grade purity, obtained from commercial sources. Methods. Absorption spectra were recorded from 230 nm nm in a spectrophotometer SPECORD UV VIS, Carl Zeiss, Jena (FRG), in thermostated cuvette holders at 250. Concentration of reactants was determined from their molar extinction coefficients at ph 7.0 (M~cm-1): NAD at 260 nm, NADH 6200 at 340 nm [6] and DACA at 400 nm [9]; the concentration of CAA was determined enzymatically [6]. Enzyme reaction rates in the reverse direction, reduction of aldehydes, were determined from initial linear phase of reaction progress curves, in 0.1 M sodium phosphate buffer ph or in 0.1 M sodium pyrophosphate buffer ph , supplemented with 0.5 mm EDTA. Initial rate data were fitted to the following equation, with the SEQUEN Fortran program of Cleland [10]: V2[Q][P] v0/eo... ( 1 ) KjqKp + Kp[Q] + Kq[P] + [Q][P] where vo is the initial rate (M.s-~), eo the concentration of enzyme active sites (M), V; the maximal catalytic constant (s-~), Kq and Kp the Michaelis constants for NADH and aldehydes (M), I~q the inhibitory constant for NADH (M), and [Q] and [P] the concentration of NADH and aldehydes (M), according to the nomenclature of Cleland [11]. Bimolecular rate constants in Figure 2 were fitted to following equations, with HBBELL and WAVL Fortran programs of Cleland [10]: C log y = log (... ) ( 2 ) 1 + Kd[H +] YL + YH(Kd[H+]) log y = log( ) ( 3 ) 1 + Kd[H where y is the bimolecular rate constant V~lKb, C and YL are ph-independent values of y in the acid, and K8 the acid dissociation constant. 366
3 RESULTS AND DISCUSSION Steady-state kinetics with DACA. aqueous solutions, from ph 6 - elaine. aldehyde [6], yeast enzyme. DACA was stable for several hours in diluted 10, and was not reduced by NADH in the absence of In a critical test designed to measure enzymatically the concentration of DACA was quantitatively reduced by excess NADH in the presence of NADH in the presence of YADH, substrates at ph 7.0. Figure 1 shows the double reciprocal plot for reduction of DACA with obtained by varying the concentration levels of both The steady-state kinetic constants for this enzymatic reaction were calculated by statistical methods (eqn 1) from data in the figure and presented in Table 1. Standard errors for kinetic constants were relatively large, because we were forced to work with subsaturating concentrations of DACA, as its solubility in water was limited to t p.m at 25~ Kinetic constants for DACA and NDMA, two chromophoric substrates of yeast en- zyme, are compared in Table 1. NDMA is a better substrate, with a specificity con- stant for aldehyde (V2/Kp) 20-fold higher, compared to DACA. Addition of substrates INTERCEPT(sI(ol SLOPE(mH.slIA) IIV 300 /..J,Is) Figure I. 1 ~ ~ 100 ~ I i i I 0 IO I IDACA ( mm'll I II NADH (ram-i) Primary double reciprocal plot for the oxidation of NADH with DACA at ph 7.0. Concentration of reactants: DACA, 24.2 p.m ( 48.5 p.m (13), 72.7 pm (A), 96.1 pm (o) and 130 pm (x); enzyme 1.23 pm. Inset represents the replot of slopes and intercepts from the primary graph [12]. 367
4 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL Table 1. Comparison of steady-state kinetic constants for reduction of various aldehydes by NADH in the presence of YADH. Substrate DACA N DMA a CAA Proplon- b aldehyde ph V2 s I Kq p.m K~q /~M Kp mm V21Kq mm-~s V~lKp mm-ls -~ V21~q/Kq s Ksq c pm Data taken from Leskovac et at. [13]. b Data taken from Leskovac et at. [2]. c Keq = [NADH][aldehyde][H+] / [NAD with lower aliphatic aldehydes is strictly ordered from ph 6-10 [2]; since V2, V2/Kq, V;/Kp and V;K~qlKq constants are much lower with DACA compared to proplonaldehyde, it appears that the order of addition of substrates with DACA is random. However, the exact extent of randomness still remains to be established [11,14]. Figure 2 shows the ph-profile of the bimolecular rate constant V21Kp for DACA, from ph ; the profile fits a dissociation curve of a single monobasic acid with pka = and a ph-independent plateau on the acid side of 0.27_+0.01 mm-ls -1. Since the enzyme was nearly saturated with NADH, the ph-dependence of V2/Kp reflects the ionization in the enzyme.nadh complex [t5]. The pk, value of 8.0 found in Figure 2 is in agreement with our recently published conclusion [t6] that this pka value reflects the ionization of His.51 in the hydrogen bonded relay system of yeast enzyme, His NADH... Thr HOH... Zn stretching from His-51 on the surface of enzyme to the active site zinc atom [1,16]. 368
5 '~' z 0.5" E =2-1.0 N Y 8 9 ph F/gure 2. ph-profile of the bimolecular rate constant V2/Kb. Upper line: CAA (o) (0.5 ram) was reduced by excess NADH (1,0 ram) in the presence of enzyme (1.5 pm). Lower line: (o) DACA (78 FM) was reduced with NADH (1.04 ram) and YADH (1,1 tim); or (A) DACA (39 pm) was reduced with NADH (0.87 mm) and YADH (2.5 I-LM). Chemical reaction of DACA with YADH. DACA (4t t~m) reacts with Tris.HCI (0.375 M) at ph 8.1 to form a Schiff base, which is evident from the gradual increase of absorbancy at 470 nm and decrease at 400 nm (Figure 3). With the aid of Guggenheim's method [17] it was found that changes in absorbancy obey the pseudo first-order rate law with a rate constant of M-~s'~; since the spectral changes have two isosbestic points, it was concluded that a single product was formed, a Schiff base between DACA and Tris base. Similar reaction rates were reported for the formation of Schiff base of glyceraldehyde-3-phosphate (0.1 Mts ~, ph 8.5) and aspartic semiaidehyde (0.2 M~s 1, ph 7.6)with Tris base [18] YADH (22 F.M) in 0.1 M sodium pyrophosphate buffer, ph 9.0, develops gradually the same spectral change as in Figure 3, in the presence of DACA (60 FM); since the activity of enzyme remains unchanged, it was concluded that only nonessential amino groups on enzyme form Schiff base with DACA. Formation of Schiff base is slow and can not influence kinetic measurements with enzyme reported above. 369
6 A 0.2 4?0 nm nm Figure 3. Difference spectrum between DACA (4t p.m) in 0.1 M sodium phosphate buffer, ph 8.1 (reference cuvette) and DACA (41 pm) in M TrisHCI buffer, ph 8.1 (sample cuvette), recorded 20 rain alter mixing DACA with Tris; light path 1 cm. Steady-state kinetics with CAA. NADH was not oxidized by CAA in the absence of enzyme. In a critical test designed to measure enzymatically the concentration of aldehyde [6], CAA was quantitatively reduced by excess NADH in the presence of yeast enzyme. Figure 4 shows the double reciprocal plot for the enzymatic reduction of CAA by NADH, obtained in a similar fashion as in Figure 1. The steady-state kinetic constants for this enzymatic reaction were calculated by statistical methods from data in the figure, and presented in Table 1. CAA is an excellent substrate of YADH, very similar to propionaldehyde. Similarity of kinetic constants for both aldehydes indicates that the kinetic mechanism with CAA may be strictly ordered, in the same way as it was reported for propionaldehyde [2]. Figure 2 shows the ph-profile of the bimolecular rate constant V2/Kp with CAA from ph ; the ph-profile fits a dissociation curve of a dibasic acid, with pka values and , and a ph-independent plateau on the acid side of mm-ls -1. Presently, we have no explanation for the difference in ph-profiles with DACA and CAA in Figure 2. 2-Chloroethanol is not a substrate of yeast enzyme. Commercial CE was oxidized 1% in a semicarbazide test [6], indicating the presence of alcohol impurity in a commercial preparation; CE, itself, was not oxidized by NAD+ in the presence of yeast enzyme. Therefore, YADH-catalyzed reaction CAA + NADH + H+ ~ CE + NAD+, must be regarded as virtually irreversible. 370
7 Chemical reaction of CAA with 'CAD/-/. YADH (0.7 pm) was readily inhibited in the presence of CAP, (350 mm) at ph 6.7 (data not shown); inhibition of yeast enzyme was a first-order process with respect to enzyme, and was characterized by a bimolecular rate constant of Mls -1. In steady-state kinetic measurements presented in Figures 2 and 4, concentrations of enzyme and CAP, were several orders of magnitude lower and the inhibition could not possibly influence the accuracy of kinetic data. It was previously reported in the literature that YADH is inhibited by bromoacetamide (ko = 0.37 M-is -1, ph 7.9, 25 ~ and iodoacetamide (ko= 0.43 Mls -1, ph 7.6, 25 ~ [19], an inhibition process considerably faster than that with CAP,. Polymerization of CAA. Commercial preparation of CAA (50 wt. % in water) is acidic and stable for a long time. Absorption spectrum of CAP, changes rapidly in the alkaline, indicating spontaneous chemical changes of CAA in aqueous alkaline solutions; these changes are characterized by increase in absorbancy at 240 nm and 6 E 5 z 15 ICAA(mH'I).J J u,l ;e 1 t /NADH (mh "1) Figure 4. Primary double reciprocal plot for the oxidation of NADH with CAP, at ph 9. Concentration of reactants: CAA, 0.72 mm (.), mm ([~), 1.77 mm (A) and 3.46 mm (o); enzyme 25.5 nm. Inset represents the replot of slopes and intercepts from the primary graph [12]. 371
8 0,6 Z,< 0,4 13,2 A 1,5 268nm,$ 1,0 'R 0,5 I I I I I 1, TIME (min) ph Figure 5. Spontaneous chemical changes of CAA in alkaline, at 25 ~ (A) Commercial CAA was dissolved (350 ram) in 10 mm NaOH, ph 1t.8, and spectral changes recorded at 240 nm and 268 nm, respectively; light path 1 cm. (B) Commercial CAP, was dissolved (350 mm) in phosphate buffers of increasing ph, and initial rates of increase in absorbancy at 240 nm measured at each ph value; Q = the ratio of rates at each ph value vs rate at ph nm (Figure 5A). At ph 11.8, increase in absorbancy at 240 nm was extremely rapid and was followed by a much slower increase at 268 nm (Figure 5A), suggesting a rapid and reversible formation of an intermediate from CAA, followed by a slow and irreversible transformation of intermediate into a final product [20] (eqn 4). CAA + X - - Intermediate--> Final product ( 4 ) The initial rate of formation of intermediate may be measured below ph 9; since it increases linearly with increasing concentration of hydroxyl ions, it is evident that it is base-catalyzed (Figure 5B). Spontaneous spectral changes of CAA were ascribed to its polymerization in alkaline; this spontaneous polymerization at ph 7-9 was too slow to influence enzyme kinetic data shown in Figures 2 and 4, in any way. Conc/usions. DACA is the first chromophoric carbonyl substrate of yeast alcohol dehydrogenase reported, so far, In the literature. Since DACA Is stable in aqueous buffers, it may become useful in analytical biochemistry. YADH-catalyzed reaction CAA + NADH + H --> CE + NAD* is virtually irreversible, and it is the first irreversible YADH-catalyzed enzymatic reaction reported, so far, in the literature. Therefore, 372
9 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL chioroacetaldehyde may find an application in biotechnology and chemoenzyme syn- theses whenever an irreversible removal of substrates is required. ACKNOWLEDGM ENTS This work was supported by research grants from the Ministry of Science and Technology, Republic of Serbia, Yugoslavia. LITERATURE 1. Eklund, H. and Branden, C.-t. (1987) in Biological Macromolecules and Assemblies (Jurnak, F. and McPherson, A., Eds.), Vol. 3, pp , Wiley, New York. 2. Leskovac, V., Trivi(~, S. and Anderson, B. M. (1996) Indian J. Biochem. Biophys., 33, Trivi6, S. and Leskovac, V. (1994) Indian J. Biochem. Biophys., 31, Weinhold, E. G. and Benner, $. A. (1995) Protein Engng., 8, Klinman, J. P. (1972) J. Biol. Chem., 247, Bergmeyer, H. U. (1970) Methoden der enzymatischen Analyse, Verlag Chemie, WeinheimlBergstrasse., t7. t8. t Hayes, J. E. and Velick, S. F. (1954) J. Biol. Chem., 207, Leskovac, V., Trivi6, S. and Panteli6, M. (1993) Anal. Biochem., 2146, Dunn, M. F. and Hutchinson, J. S. (1973) Biochemistry, 12, Cleland, W. W. (1979) Methods Enzymol., 63, Cleland, W. W. (1970) in The Enzymes (Boyer, P. D., Ed.), lind Ed., Vol. II, pp. 1-64, Academic Press, New York. Segel, I. H. (1975) Enzyme Kinetics, pp , Wiley, New York. Leskovac, V., Trivi6, S. and Anderson, B.M. (t996) Italian J. Biochem, 45, Dalziel, K. (1975) in The Enzymes (Boyer, P. D., Ed.), IIIrd. Ed., Vol. XL, pp. 1-60, Academic Press, New York. Fersht, A. (1985) El~/me structure and mechanism, pp , II Ed., W. H. Freeman, New York. Leskovac, V., Trivi6, S. and Anderson, B. M. (1997) Int. J. Biochem. Cell Biol., 25, Guggenheim, E. A. (1926) Phil. Mag., 2, Ogilvie, J. W. and Whitaker, S. C. (1976) Biochim. Biophys. Acta, 445, Piapp, B. V. (1982) Methods Enzymol., 87, Plowman, K. M. (1972) Enzyme Kinetics, pp. 7-11, McGraw-Hill, New York. 373
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