/ . Biochem., 79, 543-548 (1976)
Competitive Inhibition of Hexokinase Isoenzymes by Mercurials Fusae KANDA, Toshie KAMIKASHI, and Sadahiko ISHIBASHI Department of Physiological Chemistry, Hiroshima University School of Medicine, Hiroshima, Hiroshima 734 Received for publication, September 2, 1975
A difference in the mode of inhibition of hexokinase [EC 2.7.1.1] isoenzymes by />-chloromercuribenzenesulfonate was confirmed with respect to glucose between two Type I isoenzyme preparations purified from the kidney and spleen of rat. Essentially the same difference was observed when galactose was used as the substrate in place of glucose, as the kidney Type I isoenzyme was inhibited in a competitive manner while the spleen counterpart was inhibited in a non-competitive manner by sulfhydryl inhibitor. Both the Type I isoenzymes, however, were competitively inhibited by other mercurial sulfhydryl inhibitors, methyl and butyl mercuric chlorides. On the other hand, the Type II hexokinase isoenzymes purified from the muscle, heart, and spleen were all inhibited competitively by />-chloromercuribenzenesulfonate with respect to glucose. The mechanism of competitive inhibition of the hexokinase isoenzymes by sulfhydryl inhibitors was discussed in view of the difference in the mode of action of the mercurials with different isoenzymes.
Hexokinase [ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1] isoenzymes in mammalian tissues have generally been classified into four types, mainly according to the electrophoretic mobility and chromatographic behavior (7, 2). These types were found to have similar Km values for glucose and ATP irrespective of the source tissue (3). In previous studies, however, we observed that hexokinase Type I isoenzymes from various rat tissues differed markedly from each other in sensitivity to pchloromercuribenzenesulfonate (abbreviated as PCMBS) (4), and that the mode of inhibition by the sulfhydryl inhibitor was also different between isoenzymes of the same type (5), though their Km values for the substrates and electrophoretic mobilities were all similar. Furthermore, in a recent study, we found that the Type I isoenzymes in the mitochondria Vol. 79, No. 3, 1976
and cytosol of the same tissue, brain, differed from each other in mode of inhibition (6). In the latter two studies, it was found that PCMBS inhibited some of the Type I isoenzymes competitively with respect to both glucose and ATP. It seems strange, however, that such a sulfhydryl inhibitor acts in a competitive manner, though this type of inhibition by sulfhydryl inhibitors has been reported for a number of enzymes (7), including a report by Parry and Walker (8) on the competitive inhibition of rat hepatic hexokinase Type IV [glucokinase ; EC 2.7.1. 2] by several sulfhydryl inhibitors with respect to glucose. To elucidate the mechanism involved in the difference in sensitivity and mode of response to the inhibitor between hexokinase isoenzymes of the same type as well as to clarify the nature of the competition, further examinations were
543
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F. KANDA, T. KAMIKASHI, and S. ISHIBASHI
undertaken with the use of other hexoses and mercurial sulfhydryl inhibitors in the present study. Hexokinase Type II isoenzymes were also purified and subjected to similar examinations. MATERIALS AND METHODS Materials—ATP, glucose-6-phosphate dehydrogenase [EC 1.1.1.49], NADP+, and PCMBS were purchased from Sigma Chemical Co. Methyl- and butylmercuric chlorides were kindly donated by Prof. N. Imura, Kitasato University. Preparation of Hexokinase Isoenzymes— Male rats of Donryu strain weighing 150—180 g were used throughout. Hexokinase Type I isoenzymes in the cytosolic fraction, obtained as the 100,000 g supernatant, were purified from the kidney and spleen by ammonium sulfate precipitation and repeated DEAE-Sephadex A50 and Sephadex G200 column chromatographies, as peported in previous papers (5, 9, 10). Preparations of kidney and spleen Type I isoenzyme with a specific activity of 6.4 and 6.7 units (^mole NADPH produced/min) per mg protein, respectively, were used in the following examinations. The Type II isoenzymes were purified from the cytosolic fraction of the femoral skeletal muscle, heart, and spleen by the same procedure, yielding preparations with specific activity of 11.3, 1.0, and 0.13 units/mg protein, respectively. Analysis of Hexokinase Activity—Hexokinase activity was measured by following the increase in the absorption at 340 nm due to the formation of NADPH coupled with glucose 6-phosphate formation, in an assay system containing 10.0 mM glucose, 3.7 mM ATP, 5.0 mM MgCU, 0.13 mM NADP+, 0.55 U/ml of glucose-6-phosphate dehydrogenase and an aliquot of the isoenzyme preparation, according to the method of Walker (77). The hexokinase isoenzyme profile was checked by electrophoresis and subsequent development on a cellulose acetate membrane (Millipore) by the method of Sato et al. (72), with some modifications (9). Protein was determined either colorimetrically (13) or fluorimetrically (14). In the inhibition studies, the concentration
of the substrate was varied in the control series as well as in the experimental series, in which the mercurial sulfhydryl inhibitor was added to the enzyme assay system just before activity determination. The concentration of the inhibitor, shown in the legends of the figures, was decided by means of a previous examination to inhibit the enzyme activity to 60—80%> of the original activity, as reported in the previous paper (5). RESULTS Further Examinations of the Experimental Conditions for Inhibition of Type I Hexokinase Activity by PCMBS—The previous results (5) were first confirmed in the present study with the newly prepared isoenzyme preparations, demonstrating that the activity of cytosolic hexokinase Type I from rat kidney was competitively inhibited by PCMBS with respect to both glucose and ATP, while the activity of the same type from the spleen was competitively inhibited with respect to ATP but non-competitively with respect to glucose. However, it was thought necessary to investigate the experimental conditions to confirm that this result was actually due to the effect of the sulfhydryl inhibitor on hexokinase. The possibility that the above results might be due to an effect of PCMBS on glucose-6-phosphate dehydrogenase could be ruled out, since the activity of the dehydrogenase was not affected by the sulfhydryl inhibitor at concentrations of 100-250 nM. The possibility of the involvement of non-specific dehydrogenase was also ruled out since no NADP+ was reduced without the addition of glucose-6-phosphate dehydrogenase to the enzyme assay system. Another possibility that competitive inhibition might be temporary and gradually replaced by the non-competitive type was also ruled out, since irreversible inactivation of the enzyme was negligible under the present experimental condition. Inhibition of Activity of Type I Hexokinase Isoenzymes with Respect to Galactose as the Substrate—Similar studies were performed with galactose in place of glucose, to see if the mode of inhibition by PCMBS depended on the struc/. Biochem.
INHIBITION OF HEXOKINASE ISOENZYMES BY MERCURIALS
ture of the hexose. The assay system was essentially the same as that with glucose, except for the replacement of glucose by galactose. As reported previously (75), galactose 6-phosphate formed by the action of hexokinase could serve as a substrate for glucose-6-phosphate dehydrogenase in the assay system, since the production of NADPH depended on the addition of the dehydrogenase and on the galactose concentration. Thus, the assay system was judged to be suitable for kinetic studies of hexokinase with respect to galactose. The Km values for galactose of the kidney and spleen Type I isoenzyme were 2.0 and 2.2 mM, respectively. The values were about 10 times those for glucose (3,5). This suggests that the hydroxyl at position 4 in the hexose molecule is involved in the action of this type of hexokinase.
(a)
545
The mode of inhibition by PCMBS of the two Type I isoenzymes with respect to galactose was the same as that observed with respect to glucose ( 5 ) . As shown in Fig. 1, the kidney Type I isoenzyme was inhibited competitively by the sulfhydryl inhibitor, while the spleen counterpart was inhibited non-competitively. The results support the previous conclusion that hexokinase isoenzyme of the same type are not identical as regards response to the sulfhydryl inhibitor and that the inhibitor affects some of the mammalian hexokinase in a competitive manner. Similar studies with mannose and 2-deoxyglucose, however, were unsuccessful as NADPH was hardly formed in the present assay system. Inhibition of Activity of Type I Hexokinase Isoenzymes by Other Organic Mercurials—To clarify the mechanism of competitive inhibition of the hexokinases by PCMBS, similar kinetic studies were carried out with methyl- and butylmercuric chlorides, which are mercurial
/ •
i/v 30 !
/ /
20-
1 2 1/GLUCOSE X10-M
-5
-3
-2
-1
0
1 2 3 4 I/GALACTOSE i ICT'M
Fig. 1. Double-reciprocal plots for the velocity of hexokinase Type I isoenzymes from (a) kidney and (b) spleen versus galactose concentration. O, control; • , with />-chloromercuribenzenesulfonate (100 nM). The velocity was measured as described in "MATERIALS AND METHODS" except that galactose was used in place of glucose, v is expressed as Vol. 79, No. 3, 1976
2 3 1/ATP XKT'M
Fig. 2. Double-reciprocal plots for the velocity of hexokinase Type I isoenzyme from kidney versus (a) glucose and (b) ATP concentrations. A, control; • , with methylmercuric chloride (60 nM). The other conditions were the same as in Fig. 1, except for the difference of substrate.
F. KANDA, T. KAMIKASHI, and S. ISHIBASHI
546
sulfhydryl inhibitors of smaller molecular size. As shown in Fig. 2, methylmercuric chloride competitively inhibited kidney Type I hexokinase activity with respect to both glucose and ATP. The activity of the spleen Type I isoenzyme, which was non-competitively inhibited by PCMBS with respect to glucose (5), was also inhibited competitively by methylmercuric chloride with respect to both substrates, as shown in Fig. 3. In other words, a difference in the mode of inhibition between the two Type I isoenzymes, as observed with PCMBS, was not observed with methylmercuric chloride. The mode of inhibition by butylmercuric chloride was essentially the same as that by methylmercuric chloride, both the kidney and spleen Type I isoenzymes being inhibited
petitively with respect to both substrates. Thus, competitive - type inhibition of hexokinases was also observed with mercurials of smaller size, though, no difference in the mode of inhibition was observed between isoenzymes of the same type. Inhibition of Activity of Type IIHexokinase Isoenzymes—Examinations were undertaken to see whether such difference between isoenzymes of the same type in the response to PCMBS and such competitive-type inhibition by mercurial sulfhydryl inhibitors as were observed in the case of Type I hexokinase isoenzymes could be observed with isoenzymes of the other type. As shown in Fig. 4, a kinetic study revealed that the Type II isoenzyme from skeletal muscle was competitively inhibited by PCMBS with respect to glucose and non-competitively with respect to ATP. The mode of inhibition of this Type II isoenzyme was different from that observed with several Type I isoenzymes (5), and similar to
IX GUKOSEx10"M
-
1
0
1
2 3 1/ATP XX)"'M
Fig. 3. Double-reciprocal plots for the velocity of hexokinase Type I isoenzyme from spleen versus (a) glucose and (b) ATP concentrations. A, control; • , with methylmercuric chloride (50 nM in (a) and 70 nM in (b)). The other conditions were the same as in Fig. 2.
1/ATPxiO"
Fig. 4. Double-reciprocal plots for the velocity of hexokinase Type II isoenzyme from muscle versus (a) glucose and (b) ATP concentrations. A, control; A, with /xhloromercuribenzenesulfonate (100 nM in (a) and 130 nM in (b)). The other conditions were the same as in Fig. 2. / . Biochem.
547
INHIBITION OF HEXOKINASE ISOENZYMES BY MERCURIALS
DISCUSSION
4 - 3 - 2 - 1
-4
0
0
1
2
4
3
4
5
8 12 1/ATP X10 M
Fig. 5. Double-reciprocal plots for the velocity of hexokinase Type II isoenzyme from heart versus (a) glucose and (b) ATP concentrations. A, control; A, with />-chloromercuribenzenesulionate (130nM in (a) and 200 nM in (b)). The other conditions were the same as in Fig. 2.
that observed with hepatic Type IV hexokinase isoenzyme by Parry and Walker (5). The heart Type II isoenzyme was also inhibited competitively by PCMBS with respect to glucose, while the mode of inhibition with respect to ATP was of mixed type, as shown in Fig. 5. The mode of inhibition of the spleen Type II isoenzyme was similar to that of the heart one. Thus, competitive - type inhibition by PCMBS was also observed in the case of hexokinase Type II isoenzymes with respect to glucose, though the inhibition with respect to ATP was not consistent. As far as the three Type II isoenzymes were concerned, no marked difference was found in the response to PCMBS, unlike the case of the Type I isoenzymes (5). Vol. 79, No. 3, 1976
The nature of the apparently competitive inhibition of hexokinase by sulfhydryl inhibitor is debatable, since it is thought that such an inhibitor forms covalent linkages with sulfhydryl groups in the enzyme molecule to effect non-competitive inhibition. Parry and Walker (
Competitive Inhibition of Hexokinase Isoenzymes by Mercurials Fusae KANDA, Toshie KAMIKASHI, and Sadahiko ISHIBASHI Department of Physiological Chemistry, Hiroshima University School of Medicine, Hiroshima, Hiroshima 734 Received for publication, September 2, 1975
A difference in the mode of inhibition of hexokinase [EC 2.7.1.1] isoenzymes by />-chloromercuribenzenesulfonate was confirmed with respect to glucose between two Type I isoenzyme preparations purified from the kidney and spleen of rat. Essentially the same difference was observed when galactose was used as the substrate in place of glucose, as the kidney Type I isoenzyme was inhibited in a competitive manner while the spleen counterpart was inhibited in a non-competitive manner by sulfhydryl inhibitor. Both the Type I isoenzymes, however, were competitively inhibited by other mercurial sulfhydryl inhibitors, methyl and butyl mercuric chlorides. On the other hand, the Type II hexokinase isoenzymes purified from the muscle, heart, and spleen were all inhibited competitively by />-chloromercuribenzenesulfonate with respect to glucose. The mechanism of competitive inhibition of the hexokinase isoenzymes by sulfhydryl inhibitors was discussed in view of the difference in the mode of action of the mercurials with different isoenzymes.
Hexokinase [ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1] isoenzymes in mammalian tissues have generally been classified into four types, mainly according to the electrophoretic mobility and chromatographic behavior (7, 2). These types were found to have similar Km values for glucose and ATP irrespective of the source tissue (3). In previous studies, however, we observed that hexokinase Type I isoenzymes from various rat tissues differed markedly from each other in sensitivity to pchloromercuribenzenesulfonate (abbreviated as PCMBS) (4), and that the mode of inhibition by the sulfhydryl inhibitor was also different between isoenzymes of the same type (5), though their Km values for the substrates and electrophoretic mobilities were all similar. Furthermore, in a recent study, we found that the Type I isoenzymes in the mitochondria Vol. 79, No. 3, 1976
and cytosol of the same tissue, brain, differed from each other in mode of inhibition (6). In the latter two studies, it was found that PCMBS inhibited some of the Type I isoenzymes competitively with respect to both glucose and ATP. It seems strange, however, that such a sulfhydryl inhibitor acts in a competitive manner, though this type of inhibition by sulfhydryl inhibitors has been reported for a number of enzymes (7), including a report by Parry and Walker (8) on the competitive inhibition of rat hepatic hexokinase Type IV [glucokinase ; EC 2.7.1. 2] by several sulfhydryl inhibitors with respect to glucose. To elucidate the mechanism involved in the difference in sensitivity and mode of response to the inhibitor between hexokinase isoenzymes of the same type as well as to clarify the nature of the competition, further examinations were
543
544
F. KANDA, T. KAMIKASHI, and S. ISHIBASHI
undertaken with the use of other hexoses and mercurial sulfhydryl inhibitors in the present study. Hexokinase Type II isoenzymes were also purified and subjected to similar examinations. MATERIALS AND METHODS Materials—ATP, glucose-6-phosphate dehydrogenase [EC 1.1.1.49], NADP+, and PCMBS were purchased from Sigma Chemical Co. Methyl- and butylmercuric chlorides were kindly donated by Prof. N. Imura, Kitasato University. Preparation of Hexokinase Isoenzymes— Male rats of Donryu strain weighing 150—180 g were used throughout. Hexokinase Type I isoenzymes in the cytosolic fraction, obtained as the 100,000 g supernatant, were purified from the kidney and spleen by ammonium sulfate precipitation and repeated DEAE-Sephadex A50 and Sephadex G200 column chromatographies, as peported in previous papers (5, 9, 10). Preparations of kidney and spleen Type I isoenzyme with a specific activity of 6.4 and 6.7 units (^mole NADPH produced/min) per mg protein, respectively, were used in the following examinations. The Type II isoenzymes were purified from the cytosolic fraction of the femoral skeletal muscle, heart, and spleen by the same procedure, yielding preparations with specific activity of 11.3, 1.0, and 0.13 units/mg protein, respectively. Analysis of Hexokinase Activity—Hexokinase activity was measured by following the increase in the absorption at 340 nm due to the formation of NADPH coupled with glucose 6-phosphate formation, in an assay system containing 10.0 mM glucose, 3.7 mM ATP, 5.0 mM MgCU, 0.13 mM NADP+, 0.55 U/ml of glucose-6-phosphate dehydrogenase and an aliquot of the isoenzyme preparation, according to the method of Walker (77). The hexokinase isoenzyme profile was checked by electrophoresis and subsequent development on a cellulose acetate membrane (Millipore) by the method of Sato et al. (72), with some modifications (9). Protein was determined either colorimetrically (13) or fluorimetrically (14). In the inhibition studies, the concentration
of the substrate was varied in the control series as well as in the experimental series, in which the mercurial sulfhydryl inhibitor was added to the enzyme assay system just before activity determination. The concentration of the inhibitor, shown in the legends of the figures, was decided by means of a previous examination to inhibit the enzyme activity to 60—80%> of the original activity, as reported in the previous paper (5). RESULTS Further Examinations of the Experimental Conditions for Inhibition of Type I Hexokinase Activity by PCMBS—The previous results (5) were first confirmed in the present study with the newly prepared isoenzyme preparations, demonstrating that the activity of cytosolic hexokinase Type I from rat kidney was competitively inhibited by PCMBS with respect to both glucose and ATP, while the activity of the same type from the spleen was competitively inhibited with respect to ATP but non-competitively with respect to glucose. However, it was thought necessary to investigate the experimental conditions to confirm that this result was actually due to the effect of the sulfhydryl inhibitor on hexokinase. The possibility that the above results might be due to an effect of PCMBS on glucose-6-phosphate dehydrogenase could be ruled out, since the activity of the dehydrogenase was not affected by the sulfhydryl inhibitor at concentrations of 100-250 nM. The possibility of the involvement of non-specific dehydrogenase was also ruled out since no NADP+ was reduced without the addition of glucose-6-phosphate dehydrogenase to the enzyme assay system. Another possibility that competitive inhibition might be temporary and gradually replaced by the non-competitive type was also ruled out, since irreversible inactivation of the enzyme was negligible under the present experimental condition. Inhibition of Activity of Type I Hexokinase Isoenzymes with Respect to Galactose as the Substrate—Similar studies were performed with galactose in place of glucose, to see if the mode of inhibition by PCMBS depended on the struc/. Biochem.
INHIBITION OF HEXOKINASE ISOENZYMES BY MERCURIALS
ture of the hexose. The assay system was essentially the same as that with glucose, except for the replacement of glucose by galactose. As reported previously (75), galactose 6-phosphate formed by the action of hexokinase could serve as a substrate for glucose-6-phosphate dehydrogenase in the assay system, since the production of NADPH depended on the addition of the dehydrogenase and on the galactose concentration. Thus, the assay system was judged to be suitable for kinetic studies of hexokinase with respect to galactose. The Km values for galactose of the kidney and spleen Type I isoenzyme were 2.0 and 2.2 mM, respectively. The values were about 10 times those for glucose (3,5). This suggests that the hydroxyl at position 4 in the hexose molecule is involved in the action of this type of hexokinase.
(a)
545
The mode of inhibition by PCMBS of the two Type I isoenzymes with respect to galactose was the same as that observed with respect to glucose ( 5 ) . As shown in Fig. 1, the kidney Type I isoenzyme was inhibited competitively by the sulfhydryl inhibitor, while the spleen counterpart was inhibited non-competitively. The results support the previous conclusion that hexokinase isoenzyme of the same type are not identical as regards response to the sulfhydryl inhibitor and that the inhibitor affects some of the mammalian hexokinase in a competitive manner. Similar studies with mannose and 2-deoxyglucose, however, were unsuccessful as NADPH was hardly formed in the present assay system. Inhibition of Activity of Type I Hexokinase Isoenzymes by Other Organic Mercurials—To clarify the mechanism of competitive inhibition of the hexokinases by PCMBS, similar kinetic studies were carried out with methyl- and butylmercuric chlorides, which are mercurial
/ •
i/v 30 !
/ /
20-
1 2 1/GLUCOSE X10-M
-5
-3
-2
-1
0
1 2 3 4 I/GALACTOSE i ICT'M
Fig. 1. Double-reciprocal plots for the velocity of hexokinase Type I isoenzymes from (a) kidney and (b) spleen versus galactose concentration. O, control; • , with />-chloromercuribenzenesulfonate (100 nM). The velocity was measured as described in "MATERIALS AND METHODS" except that galactose was used in place of glucose, v is expressed as Vol. 79, No. 3, 1976
2 3 1/ATP XKT'M
Fig. 2. Double-reciprocal plots for the velocity of hexokinase Type I isoenzyme from kidney versus (a) glucose and (b) ATP concentrations. A, control; • , with methylmercuric chloride (60 nM). The other conditions were the same as in Fig. 1, except for the difference of substrate.
F. KANDA, T. KAMIKASHI, and S. ISHIBASHI
546
sulfhydryl inhibitors of smaller molecular size. As shown in Fig. 2, methylmercuric chloride competitively inhibited kidney Type I hexokinase activity with respect to both glucose and ATP. The activity of the spleen Type I isoenzyme, which was non-competitively inhibited by PCMBS with respect to glucose (5), was also inhibited competitively by methylmercuric chloride with respect to both substrates, as shown in Fig. 3. In other words, a difference in the mode of inhibition between the two Type I isoenzymes, as observed with PCMBS, was not observed with methylmercuric chloride. The mode of inhibition by butylmercuric chloride was essentially the same as that by methylmercuric chloride, both the kidney and spleen Type I isoenzymes being inhibited
petitively with respect to both substrates. Thus, competitive - type inhibition of hexokinases was also observed with mercurials of smaller size, though, no difference in the mode of inhibition was observed between isoenzymes of the same type. Inhibition of Activity of Type IIHexokinase Isoenzymes—Examinations were undertaken to see whether such difference between isoenzymes of the same type in the response to PCMBS and such competitive-type inhibition by mercurial sulfhydryl inhibitors as were observed in the case of Type I hexokinase isoenzymes could be observed with isoenzymes of the other type. As shown in Fig. 4, a kinetic study revealed that the Type II isoenzyme from skeletal muscle was competitively inhibited by PCMBS with respect to glucose and non-competitively with respect to ATP. The mode of inhibition of this Type II isoenzyme was different from that observed with several Type I isoenzymes (5), and similar to
IX GUKOSEx10"M
-
1
0
1
2 3 1/ATP XX)"'M
Fig. 3. Double-reciprocal plots for the velocity of hexokinase Type I isoenzyme from spleen versus (a) glucose and (b) ATP concentrations. A, control; • , with methylmercuric chloride (50 nM in (a) and 70 nM in (b)). The other conditions were the same as in Fig. 2.
1/ATPxiO"
Fig. 4. Double-reciprocal plots for the velocity of hexokinase Type II isoenzyme from muscle versus (a) glucose and (b) ATP concentrations. A, control; A, with /xhloromercuribenzenesulfonate (100 nM in (a) and 130 nM in (b)). The other conditions were the same as in Fig. 2. / . Biochem.
547
INHIBITION OF HEXOKINASE ISOENZYMES BY MERCURIALS
DISCUSSION
4 - 3 - 2 - 1
-4
0
0
1
2
4
3
4
5
8 12 1/ATP X10 M
Fig. 5. Double-reciprocal plots for the velocity of hexokinase Type II isoenzyme from heart versus (a) glucose and (b) ATP concentrations. A, control; A, with />-chloromercuribenzenesulionate (130nM in (a) and 200 nM in (b)). The other conditions were the same as in Fig. 2.
that observed with hepatic Type IV hexokinase isoenzyme by Parry and Walker (5). The heart Type II isoenzyme was also inhibited competitively by PCMBS with respect to glucose, while the mode of inhibition with respect to ATP was of mixed type, as shown in Fig. 5. The mode of inhibition of the spleen Type II isoenzyme was similar to that of the heart one. Thus, competitive - type inhibition by PCMBS was also observed in the case of hexokinase Type II isoenzymes with respect to glucose, though the inhibition with respect to ATP was not consistent. As far as the three Type II isoenzymes were concerned, no marked difference was found in the response to PCMBS, unlike the case of the Type I isoenzymes (5). Vol. 79, No. 3, 1976
The nature of the apparently competitive inhibition of hexokinase by sulfhydryl inhibitor is debatable, since it is thought that such an inhibitor forms covalent linkages with sulfhydryl groups in the enzyme molecule to effect non-competitive inhibition. Parry and Walker (
