KINETICS OF VARIANTS OF
GLC-6-P-DEHYDROGENASE
Genetic Variants of Human Erythrocyte Glucose 6-Phosphate Dehydrogenase. I. Regulation of Activity by Oxidized and Reduced Nicotinamide-Adenine Dinucleotide Phosphate” Adeyinka Afolayant and Lucio Luzzattot
The dependence of reaction velocity on oxidized nicotinamide-adenine dinucleotide phosphate (NADP+) concentration was studied in five genetic variants of glucose 6phosphate dehydrogenase: the three common variants A, B, and A- and the two rare variants Ijebu-Ode and Ita-Bale. All these variants exhibit nonhyperbolic kinetics, with transition taking place from a state I of low affinity to a state I1 of high affinity for NADP- as the concentration of the latter is increased. The enzyme-substrate dissociation constants for the two states ( K e y K , J were determined. For the five variants, K.1 ranged from 21 to 170 IM, and KsLfrom 1.3 to 13 IM. The inhibitory effect of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) on the glucose 6-phosphate dehydrogenase reaction was studied in the three common variants and in Ijebu-Ode. The values of the inhibition constant, K,, ranged ABSTRACT:
G
lucose 6-phosphate dehydrogenase plays a key role in the metabolism of the erythrocyte as the first enzyme of the pentose phosphate pathway and the rate-limiting step for the production of NADPH.’ One of the variants of this enzyme found in human erythrocytes, designated A (for nomenclature, see World Health Organization, 1967), has a “sigmoid” saturation curve with respect to NADP+, which is markedly affected by the presence of NADPH (Luzzatto, 1967). This led us to suggest that one possible regulatory mechanism for the activity of glucose 6-phosphate dehydrogenase in human red cells is based o n its interaction with NADP+ and NADPH. Here we report studies consistent with this view, based on kinetic measurements of the interaction with NADP+ and NADPH of four other variants of glucose 6-phosphate dehydrogenase. It will be shown that genetic changes affect markedly the interaction of the enzyme with the substrate and with the product of the reaction. In particular, the saturation kinetics of one of the variants, A-, is such as to enable it to utilize the substrate much more
* From the Departments of Chemical Pathology and Haematology, University College Hospital, Ibadan, Nigeria. Receiaed M a y 25, 1970. Some of the experiments here described were included in a Ph.D. thesis (University of Ibadan) by A. A. who was supported by a grant from the Federal Government of Nigeria. This work also received financial support from the World Health Organization, and from U. S. Public Health Service Grant G M 17261. A preliminary communication on part of these data was presented at the 6th Meeting of the Federation of European Biochemical Societies, Madrid, April 1969. t Present address : Department of Biological Sciences, University of Ife, Ile-Ife, Nigeria. $ To whom to address correspondence. 1 Abbreviations used are: NADPH, reduced nicotinamide-adenine dinucleotide phosphate. NADP+, oxidized nicotinamide-adenine dinucleotide phosphate.
from 16 to 210 p ~ The . type of inhibition is not purely competitive, in that NADPH also affects the transition of the enzyme from state I to state 11. An inverse relationship between K.? and K , was found on comparing a set of four variants. This suggests that the pyridine nucleotide binding structure is different in each variant in such a way, that the closer the fit for NADP+, the looser the fit for NADPH. The findings on the “deficient” variant A- indicate that, contrary to previous views, it differs in kinetic behavior from the “normal” variants A and B, in two important respects: (a) A- has the highest affinity for NADP’; (b) Ais the least sensitive to inhibition by NADPH. Because of these properties, it is inferred that this variant will normally operate, within the intact erythrocyte, at a nearly maximal velocity, thus compensating in part for the reduced enzyme concentration.
effectively at physiological substrate concentrations. This behavior may be advantageous to the red cell and thus compensate in part for the “enzyme deficiency” associated with this variant. Materials and Methods Reagents. Glucose 6-phosphate (Boehringer und Soehne), NADP+, and NADPH (Sigma Chem. Co.) were all standardized spectrophotometrically at 340 nm. Enzyme Variants. The nomenclature followed is as recommended by World Health Organization (1967). The variants common in Nigeria A, B, and A- (Luzzatto et a / . , 1965) have been obtained from unselected blood donors, and identified by starch gel electrophoresis (Luzzatto and Afolayan, 1968). The two rare variants Ijebu-Ode and Ita-Bale (electrophoretically slower than B) have been previously described (Luzzatto and Afolayan, 1968). Purificarion. Variants A, B, and A- were partially purified from pooled blood units according to Chung and Langdon (1963a), Yoshida (1966), and Cohen and Rosemeyer (1969). The two rare variants were purified from the two original blood samples, as described in World Health Organization (1967). The specific activities (units/mg of protein) of the preparations o n which the kinetic experiments were carried out were as follows: A, 2.8; B, 1.8; A-, 0.8; Ijebu-Ode, 0.2; Ita-Bale, 0.2. All enzyme preparations were stored at -20” in 70z saturated (NHJrS04. Enzyme Kinetics. Before kinetic determinations, enzyme preparations were dialyzed a t 4” for 6-12 hr against three changes of 300 volumes of 0.05 M Tris-borate buffer (pH 8.0) containing 0.1 mM EDTA and 2 PM NADP+. Assay conditions and precautions for the determination of reaction B I O C H E M I S T R Y , VOL.
IO,
NO.
3, 1 9 7 1
415
A F O L A Y A N A N D
LUZZATTO
TABLE I : Kinetic
Parameters of Glucose 6-Phosphate Dehydrogenase Variants. NADPH Binding
NADP+ Binding J
j2-- ' j l
1
-L
T
323
20
O
;\ADP:
,JM
1: Relative reaction velocity of two common variants of glucose 6-phosphate dehydrogenase as a function of the concentration of the substrate, NADP+. The concentration of the other substrate. glucose 6-phosphate. was saturating. V,,, (expressed in L4j40imiii)was between 0 05 and 0.06. The triangles (variant B) and the full circles (variant A-) represent experimental points. The full lines are plots of eq 1, calculated for the values of parameters listed in Table I. FIGLRE
velocity at very low NADP- concentrations were previously described (Luzzatto, 1967). Culculurions. The saturation curves for each enzyme variant with respect to NADP- were analyzed according to a previously proposed model, according to which the enzyme has more than one NADP+ binding site. It was postulated (Luzzatto. 1967) that the binding of the first molecule of NADP (dissociation constant, K.,) increases the affinity for NADPof another site (dissociation constant, K,
