hfarcli 12, 1963
C o p y . i g h l 195.9 b y the Amerrran Chemtca. Sortely
Studies on the Mechanism of Enzyme-Catalyzed Oxidation-Reduction Reactions. VI.* Kinetic Studies with Yeast L( +)-Lactate Dehydrogenase J. w. H I N K s o N t
AND
H. R. MAHLER$
From the Department of Chemistry, Indiana University, Bloomington, Indiana Received May 25, 1962
The kinetics of the reaction catalyzed by crystalline lactate dehydrogenase of yeast have been investigated in a sodium pyrophosphate-acetic acid buffer a t pH 7.5 and 20", with L-lactate as the substrate and ferricyanide, cytochrome c. or 2,6-dichlorophenolindophenolas the acceptor. The observed rate-law is of the form = 00 $1 ' S + $2 ' A q12 ' S A P(mo' Q,' 'S +*','A A- +12'/SAj, where E,, S, A , and P are the stoichiometric concentrations in moles liter of enzyme, lactate, acceptor, and pyruvate, and the 6's are empirical parameters. With relatively concentrated ferricyanide (0.24-0.97 mM) the reaction is shown to be consistent with the kiS k3A k, hi kyA following mechanism: E ES EXY E Z -+ EZ' A,,,,; EZ' ---j E A,c,,; k, kr 1 0 ksP k,iP E E P . With indophenol as acceptor the above mechanism is satisfied but with k s Y 0.
-
--
+
+
-.
+
-ki:
-
+
+
+
+
With ferricyamde a t low concentration and with cytochrome c a different mechanism appears k,S ks k A k;A ksP to be involved: E +E S --+ EX P; EX --+E Y Are$;E Y E t Arrd; E EP. kii kr The dissociation constants for the enzyme-substrate complex have been calculated for all these cases and were found to be acceptor-independent and equal to -6 >; lo-* M.
-
+
+
Yeast L: j-lactate dehydrogenase, commonly referred'to as cytochrome b?. was first crystallized by Appleby and Morton (1954,1959a J,in whose laboratories many of the properties of the enzyme were characterized (Appleby and Morton, 1959a,b, 1960; Appleby et al., 1960; Armstrong et ai., 1960; Morton et al., 1961; Morton and Armstrong, 1961; Morton, 1961 . In an effort to learn more about the mechanism of action of this enzyme which possesses both riboflavin5 '-monophosphate and protoheme as prosthetic groups (Appleby and Morton, 1954, 195913; Boeri et al., 19551. the kinetics of the enzyme-catalyzed reaction have been investigated in some detail. These kinetic studies were inaugurated ( a ) to decide whether during an enzyme-catalyzed reaction employing the same oxidative enzyme a different sequence of reactions must be postulated when different acceptors are used to oxidize the substrate, (bi to decide whether this oxidation pro-
* For paper V of
this series see Fernandez et al. 11962,.
--
ceeds uia a ternary complex, ( c ) to obtain some indication concerning the numerical vaiues of certain critical equilibrium and rate constants, and ( d ) if possible to choose between or eliminate previously postulated sequences of reactions postulated for the enzymecatalyzed reaction (Morton et al., 1961; Morton and Armstrong, 1961). Such a study of this particular enzyme seemed desirable, since kinetic and mechanistic parameters established in this instance could have implications for other flavoenzymes and even serve as a soluble model system for the particulate enzymes involved in electron transport. As a basis for these kinetic studies the procedure developed by Dalziel (19573 for two-substrate systems was utilized for an analysis of the data. I t has also proved helpful to study the effects of added product upon the kinetic parameters, as suggested by Alberty (1958). With the use of Dalziel's notation the following modified general rate equation may be formulated as shown in equation (l,,where E : = total molarity of
t Predoctoral Fellow of the National Cancer Institute of
the National Institutes of Health, Bethesda, Md. Present address: Department of Physiological Chemistry, University of Minnesota, Minneapolis 14, Minn. The major part of the experimental work is taken from a dissertation submitted by J. W. Hinkson to the Graduatp School of Indiana University in partial fulfillment for the Ph.D. degree. $Supported by Grant No. G-8959 o f the National Science Foundation.
the enzyme; u0 = initial velocity of the enzymecatalyzed reaction: C' = empirical constants, generated by combinations of particular rate constants characteristic of any postulated mechanism; S = 209
210
Biochemistry
J. W. HINKSON AND H. R. MAHLER
2oor- r :
I00 .'O'
P
-
I
/
I
40-
I
/4-
20 -,.A. lr
FIG. 1.-Lineweaver-Burk plot for a wide range of DLlactate concentrations. Sodium DL-laCtate was varied from 8.3 X 10-6 M to 5.35 X lo-* M. Potassium ferricyanide was used as the acceptor and was 6.7 X lo-* m. All experiments were done in sodium pyrophosphateacetic buffer (260 pmoles in 3.0 ml) and at 20".
substrate concentration in molesjliter; A = concentration of second substrate or acceptor in moles/ liter; P = concentration of the product formed from S in moles liter. Initially, when P = 0, equation (1) simplifies to the simple Dalziel equation since all terms multiplied by P vanish. Morton's group (Morton et al., 1961; Morton and Armstrong, 1961) have previously shown that +12 is equal to zero when cytochrome c is used as acceptor and has a finite and positive value when ferricyanide is used. The present investigations corroborate and extend their findings.
of the lactate concentration in the solution. This dilution allowed a greater range of lactate concentrations to be UBed in these studies. Reaction Velocity Detenninations.-All velocity determinations were performed a t room temperature, in an air-conditioned mom controlled to a temperature of about 20°, and performed spectrophotometrically by means of a Cary Model 11recording spectrophotometer equipped with a slide wire suitable for the absorbancy range 04.1. Pliimpers (polyethylene enzyme spoons) were a g i f t of California Corporation for Biochemical Research. All reagents were placed into a l.0-cm light path absorption cell and brought to a total volume of 3 ml. The reaction was initiated by the addition of 5-25 p1 of enzyme solution stirred into the reaction mixture with either a polyethylene or a lucite enzyme spoon (see Baker, 1960, for details of construction). All calculations involving spectrophotometric determinations utilized the following m~ extinction coefficients: 1.04 a t 420 m p for ferricyanide (Appleby and Morton, 1959a), 8.9 a t 550 mp for oxidized cytochrome c, 29.9 at 550 mp for reduced cytochrome c, 21 at 550 mp for the differential extinction coefficient (reduced minus oxidized) for cytochrome c (Massey, 1959),38.8 a t 557 mp and 232 a t 424 mp for reduced YLDH,' 199 a t 265 mp for YLDH, 23.9 a t 557 mp for the differential extinction coefficient of YLDH (Appleby and Morton, 1959b), and 21 at 600 mp for 2,6-dichlorophenolindophenol (Steyn-Parve and Beinert, 1958). Graphic Determinations.-All + values were graphically determined by the method of Dalziel (1957) and represent the averages of the duplicate values obtained by that method. The +o and 9]values in the duplicate determinations with ferricyanide as acceptor (0.102 mM-1.02 mM) were within +3% and *9%, respectively, of the average values. +z values varied from +26% to 170% of the average values, while duplicate values of +12 ranged from =t5% to &30% of the average. When the acceptor was cytochrome c, $1 and +2 were within *5% and +o was within +30% of the average values.
EXPERIMENTAL PROCEDURE Reagents.-Horse heart cytochrome c was obtained from Sigma Chemical Co., St. Louis, Mo. The calcium salt of L(+)-lactate was obtained from the California Corporation for Biochemical Research, Los Angeles. After removal of the calcium by precipitation with sodium pyrophosphate the pH of the resulting solution was adjusted to the desired value with either 0.1 M acetic acid or 0.1 M sodium pyrophosphate. The disodium salt of ethylenediaminetetraacetate (EDTA, Versene) was white-label-grade material from Eastman Organic Chemicals Division, Distillation Produds Industries, Rochester, N. Y . Practical-grade acetone was redistilled over potassium permanganate. The fraction boiling between 55" and 56" was collected and used for the enzyme preparation. n-Butanol was redistillctl from practical-grade butanol, and the fraction boiling from 115-117" was utilized for the enzyme preparktion. Sodium pyruvate was C.P. grade from Schbmrz Laboratories, Inc., Mt. Vernon, N. Y. All other reagents were reagent-grade materials available from commercial sources. Crystalline yeast L I +)-lactate dehydrogenase was prepared from airdried Red Star baker's yeast according to the procedure given by Appleby and Morton (1959a). Unless stated otherwise, stock enzyme solutions which were stored in 0.5 M DL-lactate under nitrogen a t -20" were utilized for all experiments. Prior to use, the enzyme solution was frequently diluted 20- to 50-fold into 0.1 M pyrophosphate-acetate buffer, pH 7.5, to reduce the level
RESULTS Kinetics of Reaction with Ferricyanide as Acceptor.The most extensive investigations were performed when potassium ferricyanide was employed as the final electron acceptor. As seen from Figure 1 the reciprocal of the velocity of the enzyme-catalyzed reaction as a function of the reciprocal substrate concentration is linear over a wide range (8.3 X 10-SMto 5.35 X IO-") of lactate concentrations. Although not evident from the plot shown in Figure 1, there is a slight substrate inhibition a t the highest lactate concentrations used. When the reciprocal velocity of the enzyme-catalyzed reaction is plotted as a function of the reciprocal of the ferricyanide concentration (Fig. 2), a biphasic curve is obtained. This suggests the possibility that two different mechanisms are operating which depend upon the concentration of ferricyanide employed as the acceptor. A further indication that two different mechanisms may be operating was obtained after determination of the Dalziel kinetic parameters. As shown in Table I, +12 has vastly different values which depend upon the concentration range of acceptor employed. In the more concentrated ranges of ferricya1 The following abbreviations will be used in this paper: YLDH, crystalline yeast L I )-lactate dehydrogenase; EDTA, ethylenediaminetetraacetate; OD, optical density; TN, pmoles acceptor reduced per minute per mole of enzyme bound heme.
+
Vol. 2, NO.2, Mar.-Apr., 1963 I
I
211
MECHANISM OF LACTIC DEHYDROGENASE
.
r 4
11-
16 14
I /
3
12 \
::IO
-'-L 00
80
40
d d
B
a
120 160 200 240 280 320 360 [PYRUVATE] x IO3(!)
'
xlO-Z(g-'l
[Fe(CN)i3]
FIG.2.-Lineweaver-Burk plot as ferricyanide is varied. The reaction mixture contained 250 pmoles of sodium pyrophosphate-acetic acid buffer and 12 pmoles of sodium DLlactate at p H 7.5 in a total volume of 3.0 ml. The temperature was 20'.
FIG.3.-Effects of pyruvate on reaction velocity. The reaction mixture contained 12 pmoles of sodium lactate, 2.0 pmoles of potassium ferricyanide, and 250 pmoles of sodium pyrophosphate-acetic acid buffer, pH 7.5, in a total volume of 3.0 ml.
nide (0.102 mM to 1.02 mM; see experiments 1-3) has a finite positive value, while with the more dilute concentrations (6.03 X 10-2 mM to 0.24 mM; see experiment 4) + 1 2 is virtually equal to zero. Thus, with one acceptor two different mechanisms may be operating. TABLE I COMPARISON OF DALZIEL KINETICPARAMETERS AT p H 7.Fja (ferricyanide as acceptor j Expt. No. 1 2 3" 4
Conc. of Ferricyanide (mM)
0 102-1 02
0 102-1 02 0 102-1 02 006-024
on isec. x 104) 6 6 6 7
2 6 0 2
41 \M
x
42
sec. (M sec. 106) X10')
4 8 4 2
4 6 6 6
3 6 2 1
3 6 5 3
$12
(?d2
x
sec.
lolo)
Uncertain 0 66 2 4
