Yeast Hexokinase Research Articles

Comments on the kinetics and mechanism of yeast hexokinase action. Is the binding sequence of substrates to the enzyme ordered or random?

The mechanism of action of yeast hexokinase was reinvestigated in light of a recent report in which it was suggested that substrates add to the enzyme in an ordered manner with the binding of glucose required to precede the binding of ATP. Initial velocity experiments were undertaken with AMP which is a competitive inhibitor for ATP, and with the product inhibitor glucose‐6‐phosphate. The results of these studies serve to exclude the ordered mechanism for yeast hexokinase from consideration. A discussion is presented in an attempt to show that yeast hexokinase may react in a random fashion with its substrates before forming a ternary complex of enzyme‐ATP‐glucose.

A spectrophotometric assay for cellobiase

Chemical methods for measuring cellobiase activity are based on increased reducing capacity, following conversion of cellobiose into glucose (1–3). The chemical methods are time consuming, of low sensitivity, and nonspecific. Enzymic methods use glucose oxidase to estimate the amount of glucose liberated (4,5). Although more specific and sensitive, the enzymic methods are also time consuming. The two-step method requires two successive incubations, which take several hours (4). Although very sensitive, the one-step method requires 75 min for a preincubation, a highly purified glucose oxidase with negligible disaccharidase activity, and an absolutely glucose-free substrate (5). In the method for assaying cellobiase described in this paper, glucose is measured enzymically by formation of NADPH with a coupling system using yeast hexokinase, glucose-6-P dehydrogenase, and NADP, similar to previous methods involving TPN (6). The highly reproducible assay evaluated here requires only 15 min and all reagents and auxiliary enzymes of adequate quality are available commercially.

Effect of alcohol on yeast hexokinase.

The effect of alcohol upon hexokinase activity has been examined and it has been found that in the concentration range of 0.015 to 1.5 M inhibition varies directly with concentration. Increasing alcohol concentration to 2.25 M resulted in less inhibition than at 1.5 M. No evidence was obtained that hexokinase activity was accelerated by use of very low concentrations (0.0015 M) alcohol.

Kinetic study of yeast hexokinase. 1. Steady-state kinetics.

Thirty-one different models corresponding to all plausible mechanisms of glucose phosphorylation conditioned by yeast hexokinase have been analysed. Comparison of these models with the results given by kinetic study of the enzyme has enabled us to prove that the catalysis corresponds to an ordered mechanism in which hexokinase binds first glucose and then the chelate MgATP2−. The latter substrate is not able, to any great extent, to form directly the first complex with the phosphotransferase. The ionic species Mg2+ and ATP4− have only a negligible affinity for the enzyme, or the enzyme-glucose complex. Glucose-6-phosphate and ADP appear in the medium following the decomposition of the enzyme-glucose-magnesium-ATP complex. This decomposition is also controlled by an ordered mechanism, MgADP- being formed first, and glucose-6-phosphate afterwards. MgADP- is able not only to fix itself on the enzyme-glucose-6-phosphate complex but also on the enzyme-glucose complex. This mechanism is in contradiction with that of Fromm. On the other hand it agrees with that postulated but not proved by Hammes and Kochavi. The rate constant values in the proposed mechanism have been calculated. They are different from those calculated by Hammes and Kochavi.

Yeast hexokinase. 3. Sulfhydryl groups and protein dissociation.

Yeast hexokinase. 3. Sulfhydryl groups and protein dissociation.

Hexose-ATP phosphotransferases: Comparative aspects

1. 1. Glucokinase activity has been measured in livers of several amphibians, reptiles and mammals. 2. 2. The enzyme behaves adaptively in rats, mice, hamsters, guinea pigs, gerbils and squirrel monkeys. 3. 3. An antibody to yeast hexokinase was prepared; it did not react with any animal hexokinases. 4. 4. An antibody to rat liver glucokinase reacted with the glucokinases from several species but did not inhibit the low-Km hexokinases of rat, other animals or yeast, suggesting fundamental structural differences between glucokinase and the hexokinases. 5. 5. A testis-specific hexokinase has been identified in the rat, gerbil and hamster.

Kinetic study of yeast hexokinase. 2. Analysis of the progress curve of the reaction.

The kinetic study, by means of an analog computer, of the formation of glucose-6-phosphate has enabled us to confirm the ordered mechanism previously proposed by the authors. The values of the velocity constants, calculated by Hammes and Kochavi, do not allow an adjustment of the theoretical curves, given by the computer, with the experimental results. On the other hand, the values previously worked out by Noat, Ricard, Borel, and Got do enable such an adjustment.

5-Keto-D-fructose: V. PHOSPHORYLATION BY YEAST HEXOKINASE

Abstract Yeast hexokinase was shown to catalyze the phosphorylation of 5-keto-d-fructose (d-threo-2,5-hexodiulose) in the presence of ATP. The product was identified as a monophosphate ester. A procedure for the preparation of this ester and some of its properties are described. Reduction of the monophosphate ester by the NADPH-5-keto-d-fructose reductase from Gluconobacter cerinus yielded fructose 1-phosphate.

Glycolytic regulation in rat liver and hepatomas

The recent development of a series of transplantable liver tumors that display a wide range of glycolytic and respiratory activity, correlated roughly with growth rate and degree of differentiation, provides a useful tool for studies of control mechanisms in glycolysis and respiration. Experiments here described point to major sites of regulation at pyruvate kinase and/or P glycerate kinase. This assumption is based on experiments with a model system in which whole, fortified homogenates were incubated with fructose-diP as substrate and 2-deoxyglucose, P i , and yeast hexokinase were added to regenerate ADP from ATP formed by glycolysis and respiration. Measurement of O 2 uptake, lactate formation and 2-DG disappearance allowed estimation of glycolysis, respiration, and glycolytic and respiratory phosphorylation. Endogenous respiration was high in well-differentiated and low in poorly-differentiated tumors, and in both types P/O ratios were 1 to 2. Addition of FDP to homogenates of well-differentiated tumors caused only low lactate formation and low glycolytic phosphorylation and no change in respiration or respiratory ATP production. In contrast, FDP addition to poorly-differentiated tumor homogenates caused high glycolysis; and though respiration was not lowered, oxidative phosphorylation was negligible, whereas glycolytic phosphorylation was very high. These findings point to the transphosphorylating enzymes as a major site of glycolytic control, presumably owing to competition for ADP with the respiratory phosphorylation system. Additional support for this hypothesis was obtained by intermixing mitochondrial and supernatant fractions. Replacement of particles from a low-respiring, poorly-differentiated tumor by particles from a high-respiring, well-differentiated tumor resulted in a pronounced Pasteur effect; respiration was increased, together with respiratory ATP production, while glycolysis was markedly decreased. However, when particles of a high-respiring tumor were replaced with particles of a low-respiring tumor, respiration and respiratory phosphorylation were decreased, and glycolysis was markedly increased. Further evidence for glycolytic control at pyruvate kinase was obtained from enzyme assays. Pyruvate kinase levels in the poorly-differentiated tumors were up to 20-fold higher in poorly-differentiated than in well-differentiated tumors. Rat liver has two pyruvate kinase isozymes differing in adsorption on DEAE-cellulose. The low levels in liver and well-differentiated tumor predominate in the adsorbable form whereas the high levels in the poorly-differentiated tumors consist primarily of the non-adsorbed form. If competition for ADP plays a part in regulating glycolysis in liver tumors, the low levels of pyruvate kinase in the well-differentiated tumors should favor uptake of ADP by the respiratory acceptor systems and thus lower glycolysis further. On the other hand, the very high pyruvate kinase levels of the poorly-differentiated tumors should favor preferential uptake of ADP by this enzyme, to enhance glycolysis. The characteristically high glycolytic activity of tumors may in general be attributable to low levels of respiratory ADP acceptor systems in combination with high pyruvate kinase activity.

Further properties and possibel mechanism of action of adenosine 5'-triphosphate-D-glucose 6-phosphotransferase from rat liver.

1. Magnesium ions are the most effective bivalent ions in the glucokinase reaction. 2. The molecular weight of rat hepatic glucokinase is 48000-49000 as assessed by gel filtration on Sephadex G-100. 3. Anomalous kinetic behaviour at low glucose concentrations appears to be due to the formation during the purification procedure of fragments possessing modified catalytic properties, but is unlikely to be of physiological significance. 4. Extension of previous studies (Parry & Walker, 1966) suggests that glucokinase catalyses a reaction of the random Bi Bi type similar to that of yeast hexokinase. 5. The inhibitory effects of various thiol reagents suggest that a thiol group may be involved at or near the binding site of the acceptor molecule.

The correlation of resistance to 2-deoxyglucose with alkaline phosphatase levels in a human cell line

Resistance to 2DG has been studied in an established mammalian cell line. Variants having levels of alkaline phosphatase 20 to 30 times lower than the wild type were observed to possess increased sensitivity to the analogue. A correlation was observed between the level of resistance and the level of alkaline phosphatase in a number of lines of different origin. The level of resistance was found to be dependent upon the concentration of cells per volume of medium. Selection experiments demonstrated that the resistant cells exert a protective effect over the sensitive ones when the two lines are grown together in the presence of the analogue. The rate of uptake of glucose and 2-deoxyglucose from the medium was slightly higher for the resistant than for the sensitive line; the apparent hexokinase level was two fold higher in the sensitive line. Addition of purified alkaline phosphatase inhibited hexokinase activity of extracts of sensitive and resistant cells as well as that of purified commercial yeast hexokinase. Mixing of sensitive and resistant cells resulted in a slight inhibition over the values predicted on the basis of additivity. It is concluded that the mechanism of resistance to 2DG is elevated alkaline phosphatase, which acts directly on 2DG6P reconverting it to 2DG, in which state it is not toxic to the cells. This elevated alkaline phosphatase is believed to cause an apparent reduction in the level of hexokinase. The hypothesis is advanced that alkaline phosphatase can be transferred by direct cell contact, thus protecting alkaline phosphatase negative cells in mixed culture.

Hormonal control of tanning in the American cockroach—V. Some properties of the purified hormone

Purified preparations of cockroach bursicon are stable to temperatures of 55°C but are inactivated by repeated freezing and thawings. High salt concentrations and extremes in pH inhibit the tanning response. Copper and magnesium stimulate cuticular darkening but several other ions are inhibitory. Optimum temperature studies indicate maximum tanning takes place at 35° to 45°C. Bursicon activity is destroyed by short-chain alcohols at all temperatures tested. Gel filtration determinations on P-30 polyacrylamide gel and Sephadex G-50 suggest that the hormone has a molecular weight between 30,000 and 50,000. Co-chromatography of the hormone with yeast hexokinase on P-60 polyacrylamide gel indicates a molecular weight of approximately 40,000 for cockroach bursicon.

Rat Skeletal Muscle Hexokinase: II. KINETIC EVIDENCE FOR A SECOND HEXOKINASE IN MUSCLE TISSUE

The kinetics and mechanism of action of a partially purified hexokinase from rat skeletal muscle tissue were investigated. The enzyme was isolated after a series of ammonium sulfate fractionations and chromatography on diethylaminoethyl cellulose. Initial rate experiments revealed a fundamental difference between this enzyme, which is referred to as muscle hexokinase II, and another hexokinase present in muscle tissue. These observations are consistent with the demonstration by Katzen and Schimke of the existence of two hexokinase isozymes in rat skeletal muscle tissue (3). The mechanism of action of muscle hexokinase II appears to be of the rapid equilibrium random type, i.e. random addition of substrates to the enzyme, where all enzyme and substrate interactions equilibrate rapidly relative to interconversion of the central ternary complexes, enzyme-adenosine triphosphate-glucose and enzyme-adenosine diphosphateglucose 6-phosphate. The other hexokinase present in muscle tissue exhibits a reaction mechanism in which a substrate reacts with the enzyme to produce a product prior to the addition of the second substrate. Kinetic experiments with AMP, ADP, mannose, and mannose-6-P suggest that muscle hexokinase II is similar, but not identical, in its mechanism of action to yeast hexokinase, pyruvate kinase, and creatine kinase.

Yeast Hexokinase. I. Preparation of the Pure Enzyme*

Yeast Hexokinase. I. Preparation of the Pure Enzyme^*

Hormone–Enzyme Complex: Insulin–Hexokinase

CORTISONE acetate dissolved in β-lipoprotein (isolated from blood plasma of normal animals) has been shown to inhibit the activity of yeast hexokinase, while on addition of insulin the inhibition is reversed, and the activity of the enzyme restored1. It has also been found that blocking of yeast hexokinase sulphydryl groups with parachlormercuribenzoate2 or N-ethylmaleimide3 failed to affect the activity of the enzyme. Consequently, it was obvious that sulphydryl groups of hexokinase played no part in catalysis of the glucokinase reaction4.

The reaction of yeast hexokinase with sulfhydryl reagents.

Abstract The spectrophotometric assay of sulfhydryl groups by p- chloromercuribenzoate (PCMB) revealed approx. 6 SH groups per molecule of yeast hexokinase (ATP: d -hexose 6-phosphotransferase, EC 2.7.1.1). In the presence of glucose the reaction of the hexokinase with PCMB is slower. It is possible to react 3–4 SH groups at pH 7.0 in the presence of glucose without loss of activity. Under this condition an appreciable amount of activity is left after all the SH groups available for PCMB have reacted. The inactivation of the hexokinase activity by PCMB is reversible by the addition of cysteine. However, when high concentrations of the enzyme solution and correspondingly high concentrations of PCMB are incubated, the hexokinase activity is irreversibly lost. The PCMB-hexokinase complex is catalytically active but is very labile and loses its activity when glucose is not present in the solution. Sedimentation studies of the hexokinase-PCMB complex indicated that the irreversibly inactivated hexokinase consists of at least two molecular species; one sedimenting with the same velocity as the normal enzyme and the other sedimenting twice as fast.

Introduction to the Biochemistry of D-Arabinosyl Nucleosides

The chapter discusses the biochemistry of D-arabinosyl nucleosides. Arabinosyl compounds can be thought of as analogs of both ribo and deoxy ribo compounds. They do participate actively in many enzymatic reactions, e.g., deamination of ara-C and ara-A, by cytidine deaminase and adenosine deaminases, respectively, phosphorylation of ara-C, by wheat phosphotransferase, phosphorylation of cytosine arabinoside monophosphate (ara-CMP), by an appropriate kinase, the activity of adenine arabinoside triphosphate (ara-ATP), with yeast hexokinase, etc. The epimerization of the 2'-OH in the sugar does not so distort the structure, as to prevent these compounds from approaching a variety of catalytic sites in several enzymes. The apparent lack of phosphorylation of ara-M and its apparent inhibitory properties, as a nucleoside, in the conversion of cytosine monophosphate (CMP) to deoxycytidine monophosphate (dCMP) are also consistent, with the possibility that some arabinosyl compounds may not inhibit as nucleotides, but may be active solely as nucleosides. At present, there is little experimental evidence to suggest why a deficiency in deoxyribonucleotide should be lethal. The hypothesis, that in some way these deficiencies provoke chromosome breaks, is supported by the observation of these breaks in animal cells. Some slight evidence concerning deoxyribonucleic acid (DNA) degradation in bacteria, provoked by these treatments, is becoming available, of which induction of lysogenic bacteria, by such methods, may be considered to be an example. It is evident then that the chemistry and biochemistry of the D-arabinonuckosides have been launched, but that the biological activities of these compounds present numerous mysteries. The practical and esthetic aspects of the mysteries and its relation to the molecular mechanism and cellular physiology is discussed in this chapter.

Effect of Aluminum on the Uptake and Metabolism of Phosphorus by Barley Seedlings

The uptake of P(32) and its incorporation into phosphorylated compounds was examined in the roots of barley seedlings which had been pretreated with aluminum.The rate at which phosphorus increased in Al-roots was greater than in controls, especially during the first 15 minutes of incubation. It was shown that the increased phosphorus in Al-roots was P(i) and that it was almost completely exchangeable. Similar increases over controls were found when root segments were incubated in phosphorus solutions containing 10(-3)m DNP and at low temperature. The increased P(i) in Al-roots did not result in an increase in the total amount of phosphorus incorporated into phosphorylated compounds.Aluminum treatment markedly decreased the incorporation of P(32) into sugar phosphates but increased the pool size of ATP and other nucleotide triphosphates present in the roots. The specific activities of P(32) in ATP in Al-roots and controls were similar indicating that the rates of ATP synthesis were similar in each case.Preliminary investigations showed that aluminum citrate inhibited both purified yeast hexokinase and phosphorylated sugar production by crude mitochondrial extracts from barley roots.The results suggest that there are 2 reactions between aluminum and phosphorus: 1) at the cell surface or in the free space which results in the fixation of phosphate by an adsorption-precipitation reaction; 2) within the cell, possibly within the mitochondria, which results in a marked decrease in the rate of sugar phosphorylation, probably effected by the inhibition of hexokinase. The evidence does not support the view that aluminum enhances phosphorus uptake or that the superficial reaction between aluminum and phosphate interferes with phosphorus transport.

Reversible Inactivation and Dissociation of Yeast Hexokinase

Reversible Inactivation and Dissociation of Yeast Hexokinase

Adenosine Triphosphatase Activity of Yeast Hexokinase and Its Relation to the Mechanism of the Hexokinase Reaction

Adenosine Triphosphatase Activity of Yeast Hexokinase and Its Relation to the Mechanism of the Hexokinase Reaction