216
Biochemistry
J. W. HINKSON AND H. R. MAHLER
mechanisms are operating, namely, that a ternary complex is formed. ACKNOWLEDGMENTS The authors wish to acknowledge the able technical assistance of Miss Frances Oltman in this investigation. We are also deeply indebted to Dr. J. Reed of the Red Star Yeast Co., Milwaukee, Wis., who provided without charge the great quantities of cake yeast needed during this study. Appreciation is extended to Dr. R. K. Morton, who kindly supplied the authors with a preprint of ‘‘Symposium on Haematin Enzymes” prior to actual publication. REFERENCES Alberty, R. A. (1958), J. A m . Chem. Soc. 80, 1777. Appleby, C. A,, and Morton, R. K. (1954),Nature 173, 749. Appleby, C. A., and Morton, R. K. (1959a), Biochem. J. 71, 492. Appleby, C. A., and Morton, R. K. (1959b), Biochem. J. 73, 539. Appleby, C. A., and Morton, R. K. (1960), Biochem. J. 75, 258. Appleby, C. A., Morton, R. K., and Simmonds, D. H. (1960), Biochem. J. 75, 72. Armstrcng, J. M., Coates, J. H., and Morton, R. K. (1960), Nature 186, 1033. Baker, R. H., Jr. (1960), Ph.D. dissertation, Indiana University.
Baker, R. H., Jr., and Mahler, K. R. (1962), Biochemistry 1 , 35. Boeri, E., Cutolo, E., Luzzati, M., and Tosi, L. (1955), Arch. Biochem. Biophys. 56, 487. Boeri, E., and Tosi, L. (1956), Arch. Biochem. Biophys. 60, 463. Dalziel, K. (1957), Acta Chem. S c a d . 11, 1706. Fernandez, V. P., Mahler, H. R., and Shiner, V. J., Jr. (1962), Biochemistry 1 , 259. Frieden, C. (1957), J. A m . Chem. SOC.79, 1894. Guiditta, A., and Singer, T. P. (1959), J . Biol. Chem. 234, 662. Hasegawa, H., and Ogura, 0. (1961), in Haematin Enzymes (Canberra, 1959), Falk, J. H., Lemberg, R., and Morton, R. K., editors, London, Pergamon Press, p. 534. Krupka, R. M., and Laidler, K. J. (1961), J . A m . Chem. Soc. 83, 1445. Massey, V. (1959), Biochim. Biophys. Acta 34, 255. Minakami, S., Ringler, R. L., and Singer, T. P. (19621, J . Biol. Chem. 237, 569. Morton, R. K. (1961), Nature 192, 727. Morton, R. K., and Armstrong, J. M. (1961), Preprint No. 169, Symposium V, 5th Intern. Congr. of Biochem. 1. Morton, R . K., Armstrong, J. M., and Appleby, C. A. (1961), in Haematin Enzymes (Canberra, 1959), Falk, J. H., Lemberg, R., and Morton, R. K., editors, London, Pergamon Press, p. 501. Segal, H. L. (1959), in The Enzymes, vol. 1, Boyer, P. D., Lardy, H. A., and Myrback, K., editors, New York, Academic Press, Inc., p. 1. Steyn-Parve, E. P., and Beinert, H. (1958), J. Biol. Chem. 233, 845.
Studies on the Mechanism of Enzyme-Catalyzed Oxidation-Reduction Reactions. T’II.* p H Effects on the Kinetics of the Reaction Catalyzed by Yeast L(+)-Lactatc Dehydrogenase J. W. HINKsoNt AND H. R. M A H L E R ~ From the Department of Chemistry, Indiana University, Bloomington, Indiana Received May 25, 1962
The effects of pH upon the kinetics of binding of L ( +)-lactate, D ( -)-lactate, and pyruvate by crystalline yeast lada lad ate dehydrogenase have been studied by means of initial velocity measurements. These studies implicate a group on the free enzyme with a pK,’ of 6.5-7.0, which is acceptor independent and appears to be concerned with binding the groups attached to the a-carbon of the substrate and the product. The dissociation constants for the complexes of the free enzyme with L-lactate, caprylate, D-lactate, and pyruvate are 7.2 x 10-4 M, 4.2 x M, and 1.5 X lo-* M, respectively, all at pH 7.5 and 20”. 10-3 M, 7.2 x From studies of the DH dependence of an enzymecatalyzed reaction inferences k a y be made conceming the dissociation constants (pK, values) of group(s) on the free enzyme involved in the binding of the substrate and/or product. Dixon (1953a) has presented a method for evaluating such data which has been used in the present investigations. In the companion paper (Hinkson and Mahler,
1963) we have shown that the oxidation of lactate by and cytochrome ferricyanide (at concentrations 2 2 m ) c with YLDHl obeys the rate laws of equations (1) and (2), respectively,
* For paper VI of this series see Hinkson and Mahler (1963). t Predoctoral Fellow of the National Cancer Institute of the National Institutes of Health, Bethesda, Md. A major part of the work reported in this paper was taken from a dissertation submitted to the Graduate School of Indiana University in partial fulfillment of the requirements for the Ph.D. degree. Present address, Department of Physiological Chemistry, The Medical School, University of Minnesota, Minneapolis. $Supported by Grant No. G-8959 of the National Science Foundation.
where the &’s are experimentally determined parameters, interpretable in terms of mechanism-dependent specific rate constants, and E l , S , A , and P are the stoichiometric concentrat.ions of enzyme, lactate, acceptor, and pyruvate, all in moles ’liter. It appeared appropriate to determine the effect of pH on uo and
Ei/uu
=
+ $ i / S + 4 z / A + +IP/S.A + P(+o’ + + &‘/’A + diz’/S.A) E~/VO = 40 + 4 1 / s + b / A +I’/S.A
do
+l’/S
(11 (2)
The following abbreviations will be used in this paper: YLDH = crystalline yeast L( +)-lactate dehydrogenase (cytochrome b z ) ; p & = negative log,, of the kinetic parameter $=; FMN = riboflavin-5’-phosphate; OD = optical density.
Vol. 2, No. 2, Mar.-Apr., 1963
p H EFFECT3 ON YEAST LACTIC DEHYDROGENASE
217
TABLE I EFFECTS OF pH UPONTHE FOUR KINETICPARA~~ETERS
_ _ _ ___ Conditions . __-
& (M sec.) ___-
41 (M SeC.)
$v (M W.)
412
(M*W.)
Ferricyanide as Acceptor
pH pH pH pH
5.5,a no NaCl added 7.5, no NaCl added 5.5,D0.13 M NaCl 7.5, 0.13 M NaCl
pH 5.5, no NaCl added p H 7.5, no NaCl added pH 5.5, 0.13 M NaCl p H 7.5, 0.13 M NaCl
1 5 x 10-2 6 3 x 1 0 6 6 X 10-3 1 2 x 10-2
4 6 X lo-' 45x108 4 x 10-5 72x10"
Cytochrome c as Acceptor0 3.8 x 10-5
2.8 X lo-' 4 . 8 X 10-3 3.1 x 3 . 9 x 10-3
5.2 8.4 9.0
7 2 x 10-11 1.5 X 3 . 7 x 10-9 3.0 x 10-9
6.6 X 4 . 8 x 10-7 3 . 5 x 103 . 4 x 101.3 5.5 6.0 7.2
x lo-' x 10-5 x 10-6
x x x x
10-7 10-7 10-7 10-7
a The values of the parameters reported for these experiments without NaCl in the reaction mixture are an average of three determinations. Ferricyanide was varied from 0.102 mxd to 1.02 mM. lactate was varied from0.33 rrm t o 1.57 mM. The enzyme concentration for all experiments was 4.6 X lo-* M. * There is a greater error involved in the values for these parameters obtained when NaCl was present due to experimental di5culties unforeseen at the time these experiments were performed. The comparison of the values a t different PH values is, however. considered to be legitimate. L( +)-Lactate was varied from 0.33 mM t o 1.57 mM. E n z y m e c Oxidized citochrome c was varied from 4.4 m M to 44.0 mM. was 6.3 x 10 - 9 M for these experiments.
thus on the various kinetic parameters 4%. This paper presents the results of the studies, which implicate a group on the enzyme with a pK,' in the range of 6.5--7.0 in the binding of lactate. The pHdependence of the inhibition of the reaction produced by D-hCtak! and pyruvate has also been investigated, and values of the dissociation constants of the complexes between these compounds and YLDH have been determined and compared with that of the enzyme-substrate complex.
EXPERIMENTAL PROCEDURES Crystalline YLDH was prepared from air-dried Red Star baker's yeast by the method of Appleby and Morton (1959aL K i , the dissociation constants of various competitive enzyme-inhibitor complexes, was determined by the method of Dixon (1953b). Four concentrations of inhibitor and three concentrations of L( +)-lactate were used to determine K r . Thus Kr in this investigation represents an intersection of three lines in the Dixon plot. From equation ( l ) , assuming that P acts as the sole inhibitor, K , = 412)/ $11'). K,, the constant evaluated in the Dixon plot for pyruvate or any inhibitor analogous to pyruvate, is then equal to this product. If r#12 and becomes independN-H.
.O
=
! C I
H-A]
(3)
R¶
ACKNOWLEDGB~ENTS We are happy to express our thanks to the Red Star Yeast Co. (especially to Dr. J. Reed of that company), Milwaukee, Wis., who supplied the vast quantities of yeast needed for the preparation of the enzyme used in these studies. Miss Frances Oltman provided
capable technical assietance for most of the experimental work reported here. REFERENCES Alberty, R. A. (1956). J. Cell. Comp. Physiol. 47, Suppl. 1, 245. Appleby, C. A., and Morton, R. K. (1959a), Biochem. J . 71,492. Appleby, C . A., and Morton, R. K. (1959b), Biochem. J . 73,539. Bruice. T. C.. and Schmir, G. L. (1959). . .. J . A m . Chem. Soc. 81, 4552. . Bruice. T. C.. and Bruno. J. J. (1962). . , _J . Am. Chem. Soc. 84,2128. ’ Bernheim, F. (1928), B k h e m . J . 22, 1178. Boeri, E., Cutolo, E., Luzzati, M., and Tosi, L. (1955), Arch. B k h e m . Biophys. 56, 487. Boeri, E., and Tosi, L. (1956), Arch. Biochem. Biophys. 60, 463. Cerletti, P. (1959), A d . Chim. Acta 20, 243. Dikstein, S. (1959), Biochim. Biophys. Acta 36, 397. Dixon, M. (1953a), B k h e m . J . 54,457. Dixon, M. (1953b), Biwhern. J . 55,170. Edelhoch, H. (1958), J . A m . Chem. Soc. 80,6640. Erlanger, B. F. (1960), P m . Nut. Acad. Sci. U . S. 46, 1430. Hinkmn, J. W., and Mahler, H. R. (1963), Biochemistry 2, 209 (thisissue). Koshland. D. E., Jr. (1960), Adv. Enzymol. 22,58. KruDka. R. M.. and Laidler. K. J. (1960). . .. Tmns. Furadav &. 56, 1467: Lanere. N. A.. editor (1952). Handbook of Chemistrv. ed: ’8, Sandusky, Oko, Handbook Publishers, Inc., p. 1231. Lowe, H. J., and Clark, W. M. (1956), J . Biol. Chem. 221, 983. Michaelis, L., and Schwarzenbach, G. (1936), J . Biol. Chem. 123,527. Morton, R. K., Armstrong, J. M., and Appleby, C. A. (1961), in Haematin Enzymes (Canberra, 1959), Falk, J. H., Lemberg, R., and Morton, R. K., editors, London, Perganion Press.
__
Malic Dehydrogenase. V. Kinetic Studies of Substrate Inhibition by Oxalacetate* DILIPN. RAVAL AND R. G. WOLFE From the Chemistry Department, University of Oregon, Eugene Received July 9,1962
Detailed initial rate kinetic studies of substrate inhibition by oxalacetate, a t p H 8.0 in 0.05 M Tris acetate buffer, are reported. Substrate inhibition by oxalacetate occurs at concentrations above approximately 2.5 X lo-‘ Y when the DPNH concentration is 1 X lo-‘ M or less. The experimental data are consistent with a reaction mechanism involving competitive inhibition by the formation of an inactive E.oxalacetate complex, and independent uncompetitive inhibition by the formation of an E.DPN.oxalacetate complex. A previously unpublished method of evaluating kinetic parameters is presented.
Initial rate studies of a considerable number of enzymes have shown that deviations from the predictions of the Michaelis-Menten theory are surprisingly common a t relatively high substrate concentrations. Two fundamentally different types of deviation are observed. In the first type of deviation the reaction rate is apparently inhibited a t high substrate concentration. The second, less mmmonly observed deviation is that
* This investigation was supported in part by Public Health Service Research Grant No. H 3226 from the Natonal Heart Institute.
in which the reaction is apparently accelerated a t relatively high substrate concentrations. These deviations have come to be known as “substrate inhibition” and “substrate activation” respectively. Substrate activation or inhibition is observed in various types of enzymes, including hydrolases, transferases, and oxidoreductases. Moreover, the number of substrates participating in the reaction seems to have no obvious relationship to the occurrence of this anomalous behavior. For example, Wolf and Niemann (1959) have observed substrate activation of the enzyme a-chymotrypsin by the substrate methylace-
