Isolation, Characterization, and Partial Purification of a Reduced Nicotinamide Adenine Dinucleotide Phosphate-dependent Dihydroxyacetone Reductase from the Halophilic Alga Dunaliella parva

An NADP⁺-dependent dihydroxyacetone reductase, which catalyzes specifically the reduction of dihydroxyacetone to glycerol, has been isolated from the halophilic alga Dunaliella parva. The enzyme has been purified about 220-fold. It has a molecular weight of about 65,000 and is highly specific for NADPH. The pH optima for dihydroxyacetone reduction and for glycerol oxidation are 7.5 and 9.2, respectively. The enzyme has a very narrow substrate specificity and will not catalyze the reduction of glyceraldehyde or dihydroxyacetone phosphate. It is suggested that this enzyme functions physiologically as a dihydroxyacetone reductase in the path of glycerol synthesis and accumulation in Dunaliella.     

Partial purification, substrate specificity and regulation of alpha-L-glycerolphosphate dehydrogenase from Saccharomyces carlsbergensis.

alpha-L-Glycerolphosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) from Saccharomyces carlsbergensis was purified 400-fold. The enzyme preparation is free of interfering activities, such as glyceraldehyde phosphate dehydrogenase, alcohol dehydrogenase, triose phosphate isomerase and glycerolphosphatase. At pH 7.0 it is specific for NADH (Km = 0.027 mM with 0.8 mM dihydroxyacetone phosphate) and dihydroxyacetone phosphate (Km = 0.2 mM with 0.2 mM NADH). Between pH 5.0 and 6.0 the enzyme functions with NADPH, but only at 7% of the rate with NADH. Various anions (I- greater than SO42- greater than Br- greater than Cl-) act as inhibitors competing with the substrate dihydroxyacetone phosphate. Inorganic phosphate (Ki = 0.1 mM), pyrophosphate and arsenate are strong inhibitors. The nucleotides ATP and ADP are also inhibitory, but their action seems to be of the same type as the general anion competition (Ki = 0.73 mM for ATP). The results are consistent with the notion that the enzyme may regulate the redox potential of the NAD+/NADH couple during fermentation.     

Isolation of a sn-glycerol 3-phosphate: NAD oxidoreductase from spinach leaves.

An NAD-dependent glycerol 3-phosphate dehydrogenase (sn-glycerol 3-phosphate: NAD oxidoreductase; EC 1.1.1.8) has been purified from spinach leaves by a three-step procedure involving ion-exchange, gel filtration, and affinity chromatography. The enzyme has been purified over 10,000-fold to a specific activity of 38. It has a molecular weight of approximately 63,500. The pH optimum for the reduction of dihydroxyacetone phosphate is 6.8 and for glycerol 3-phosphate oxidation it is 9.5. During dihydroxyacetone phosphate reduction hyperbolic kinetics were observed when either NADH or dihydroxyacetone phosphate was the variable substrate, but concentrations of NADH greater than 150 μm were inhibitory. Michaelis constants were 0.30–0.35 mm for dihydroxyacetone phosphate and 0.01 mm for NADH. Glycerol 3-phosphate oxidation obeyed Michaelis-Menten kinetics with a K_m of 0.19 mm for NAD and 1.6 mm for glycerol 3-phosphate. The enzyme was specific for those substrates, and dihydroxyacetone, glyceraldehyde, glyceraldehyde 3-phosphate, NADPH, NADP, and glycerol were not utilized. The spinach leaf enzyme appears to be in the cytoplasm and probably functions for the production of glycerol 3-phosphate from dihydroxyacetone phosphate.     

BIOSYNTHESIS OF PHOSPHATIDIC ACID IN RAT BRAIN VIA ACYL DIHYDROXYACETONE PHOSPHATE

Abstract— The enzymes for the biosynthesis of phosphatidic acid from acyl dihydroxyacetone phosphate were shown to be present in rat brain. These enzymes were mainly localized in the microsomal fraction of 12–14 day old rat brains. The brain microsomal acyl CoA: dihydroxyacetone phosphate acyl transferase (EC 2.3.1.42), exhibited a broad pH optimum between pH 5 and 9 with maximum activity at pH 5.4. K _m for DHAP at pH 5.4 was 0.1 m m and V _max was 0.86nmol/min/mg of microsomal protein. The corresponding microsomal enzyme for the glycerophosphate pathway (acyl CoA: sn -glycerol-3-phosphate acyl transferase EC 2.3.1.15) was shown to have a different pH optimum (pH 7.6). On the basis of the differences in pH optima, differential effects of sodium cholate in the enzymes and a common substrate competition study, these acyl transferases were postulated to be two different microsomal enzymes.
Acyl DHAP:NADPH oxidoreductase (EC 1.1.1.101) in brain microsomes was found to be quite specific for NADPH as cofactor, being able to utilize NADH only at very high concentrations. This enzyme exhibited a K _m of 8.6 μ m with NADPH and V _mx of 0.81 nmol/min/mg protein. The presence of these two enzymes and the known presence of l-acyl- sn -glycerol-3-phosphate: acyl CoA acyl transferase in brain (F leming & H ajra , 1977) demonstrated the biosynthesis of phosphatidic acid in brain via acyl dihydroxyacetone phosphate. Phosphatidic acid was shown to form when dihydroxyacetone phosphate, acyl CoA, NADPH and other cofactors were incubated together with brain microsomes. Further properties of the enzymes and the probable importance of the presence of this pathway in brain were discussed.     

Alkyl dihydroxyacetone phosphate synthase in human fibroblasts and its deficiency in Zellweger syndrome

The cerebro-hepato-renal (Zellweger) syndrome is an autosomal recessive disorder biochemically characterized by the absence of morphologically distinguishable peroxisomes. Key enzymes involved in the biosynthesis of ether phospholipids, i.e., dihydroxyacetone phosphate acyltransferase and alkyl dihydroxyacetone phosphate synthase, are located in mammalian (micro)peroxisomes. We have previously shown a strikingly reduced activity of dihydroxyacetone phosphate acyltransferase in liver, brain, and cultured skin fibroblasts from Zellweger patients (Schutgens et al. 1984. Biochim. Biophys. Res. Commun. 120: 179-184). We have now extended these investigations by studying alkyl dihydroxyacetone phosphate synthase in cultured human skin fibroblasts. Enzymatic activity was determined by measuring the formation of radioactive alkyl dihydroxyacetone phosphate from palmitoyl dihydroxyacetone phosphate and [1-14C]hexadecanol as substrates. The enzyme was optimally active at pH 8.5 and was stimulated (about 2-3-fold) by the presence of 0.05% (v/v) Triton X-100. The apparent KM values for the enzyme in control fibroblasts amounted to 35 microM for palmitoyl dihydroxyacetone phosphate and 90 microM for hexadecanol. The reaction became inhibited at higher concentrations of both Triton X-100 and palmitoyl dihydroxyacetone phosphate. Control skin fibroblasts showed alkyl dihydroxyacetone phosphate synthase activity of 69 +/- 28 pmol X min-1 X mg-1 (n = 7), while fibroblasts from patients had an activity of only 6.3 +/- 1.7 pmol X min-1 X mg-1 (n = 7). Alkyl dihydroxyacetone phosphate synthase was also found to be deficient in tissue homogenates of Zellweger patients. The specific activity of this enzyme in liver, kidney, and brain homogenates from Zellweger patients was less than 15% of that in the corresponding tissues from controls.     

Kinetic study of sn-glycerol-1-phosphate dehydrogenase from the aerobic hyperthermophilic archaeon, Aeropyrum pernix K1.

A gene having high sequence homology (45-49%) with the glycerol-1-phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820). This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon. The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer. The optimum reaction temperature of this enzyme was observed to be 94-96 degrees C at near neutral pH. This enzyme was subjected to two-substrate kinetic analysis. The enzyme showed substrate specificity for NAD(P)H-dependent dihydroxyacetone phosphate reduction and NAD(+)-dependent glycerol-1-phosphate (Gro1P) oxidation. NADP(+)-dependent Gro1P oxidation was not observed with this enzyme. For the production of Gro1P in A. pernix cells, NADPH is the preferred coenzyme rather than NADH. Gro1P acted as a noncompetitive inhibitor against dihydroxyacetone phosphate and NAD(P)H. However, NAD(P)(+) acted as a competitive inhibitor against NAD(P)H and as a noncompetitive inhibitor against dihydroxyacetone phosphate. This kinetic data indicates that the catalytic reaction by glycerol- 1-phosphate dehydrogenase from A. pernix follows a ordered bi-bi mechanism.     

Isolation and characterization of crystalline methylglyoxal synthetase from Proteus vulgaris.

Methylglyoxal synthetase, which catalyzes the conversion of dihydroxyacetone phosphate to methylglyoxal and inorganic phosphate, has been isolated and crystalized in good yields from Proteus vulgaris. The enzyme was shown to be homogeneous by a variety of criteria and was found to be a dimer (Mr = 135,000; s20,w = 7.2 S) composed of two apparently identical catalytic and physical properties and their interconvertible nature suggest that they do not represent true isozymes. The enzyme is specific for dihydroxyacetone phosphate and does not form methylglyoxal from glyceraldehyde 3-phophate, glyceraldehyde, or dihydroxyacetone. Nonphosphorylated analogs are neither substrates nor competive inhibitors, but a variety of phosphorylated analogs are competitive with respect to dihydroxyacetone phosphate. The enzyme is inhibited by inorganic orthophosphate in a complex manner which is overcome by dihydroxyacetone phosphate in a signoidal manner     

Kinetic properties of a sn-glycerol-3-phosphate dehydrogenase purified from the unicellular alga Chlamydomonas reinhardtii

A sn-glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) has been purified from the unicellular green alga Chlamydomonas reinhardtii 3400-fold to a specific activity of 34 mumol/mg protein per min by a simple procedure involving two chromatographic steps on affinity dyes. The pH optimum for reduction of dihydroxyacetone phosphate was 6.8 and for glycerol 3-phosphate oxidation it was 9.5. In the direction of dihydroxyacetone phosphate reduction, the enzyme showed Michaelis-Menten kinetics. The enzyme reacted specifically with NADH and dihydroxyacetone phosphate as substrates with affinity constants of 16 and 12 microM, respectively. Product inhibition as well as competitive inhibition pattern indicated a random-bi-bi reaction mechanism for sn-glycerol-3-phosphate dehydrogenase from C. reinhardtii. The effective control of dihydroxyacetone reduction catalysed via this enzyme by ATP, Pi and NAD gave evidence for a physiological role of the enzyme in plastidic glycolysis.     

Metabolism of L-glyceraldehyde 3-phosphate in Escherichia coli.

When either 3H-labeled L-glyceraldehyde or 3H-labeled L-glyceraldehyde 3-phosphate (GAP) was added to cultures of Escherichia coli, the phosphoglycerides were labeled. More than 81% of the label appeared in the backbone of the phosphoglycerides. Chromatographic analyses of the labeled phosphoglycerides revealed that the label was normally distributed into phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. These results suggest that L-glyceraldehyde is phosphorylated and the resultant L-GAP is converted into sn-glycerol 3-phosphate (G3P) before being incorporated into the bacterial phosphoglycerides. Cell-free bacterial extracts catalyzed an NADPH-dependent reduction of L-GAP to sn-G3P. The partially purified enzyme was specific for L-GAP and recognized neither D-GAP nor dihydroxyacetone phosphate as a substrate. NADH could not replace NADPH as a coenzyme. The L-GAP:NADPH oxidoreductase had an apparent Km of 28 and 35 microM for L-GAP and NADPH, respectively. The enzyme was insensitive to sulfhydryl reagents and had a pH optimum of approximately 6.6. The phosphonic acid analog of GAP, 3-hydroxy-4-oxobutyl-1-phosphonate, was a substrate for the reductase, with an apparent Km of 280 microM.     

The purification and properties of Escherichia coli methylglyoxal synthase

1. Methylglyoxal synthase was purified over 1500-fold from glycerol-grown Escherichia coli K 12 strain CA 244. The purified enzyme was inactivated by heat or proteolysis, had a molecular weight of approx. 67000, a pH optimum of 7.5 and was specific for dihydroxyacetone phosphate with K(m) 0.47mm. 2. The possibility that a Schiff-base intermediate was involved in the reaction mechanism was investigated but not confirmed. 3. The purified enzyme lost activity, especially at low temperature, but could be stabilized by P(i). Two binding sites for P(i) may be present on the enzyme. Of other compounds tested only the substrate, dihydroxyacetone phosphate, and bovine serum albumin showed any significant stabilizing effect. 4. Phosphoenolpyruvate, 3-phosphoglycerate, PP(i) and P(i) were potent inhibitors of the enzyme. Kinetic experiments showed that PP(i) was apparently a simple competitive inhibitor, but inhibition by the other compounds was more complex. In the presence of P(i) the enzyme behaved co-operatively, with at least three binding sites for dihydroxyacetone phosphate. 5. It is proposed that methylglyoxal synthase and glyceraldehyde 3-phosphate dehydrogenase play important roles in the catabolism of the triose phosphates in E. coli. Channelling of dihydroxyacetone phosphate via methylglyoxal would not be linked to ATP formation and could be involved in the uncoupling of catabolism and anabolism.     

Survey of normal appearing mouse strain which lacks enzyme and NAD+-linked glycerol phosphate dehydrogenase: Normal pancreatic beta cell function,but abnormal metabolite pattern in skeltal muscle

We studied a mouse doubly homozygous for mutations in the genes encoding malic enzyme (EC1.1.1.40) and cytosolic glycerol phosphate dehydrogenase (EC 1.1.1.8) (cGPD). This mouse, which we call the mmgg mouse and which is the product of intercrosses between the Mod-1 mouse and the BALB/cHeA mouse, lacks activity of both enzymes. Like both parental strains the mmgg mouse is completely normal in appearance. cGPD is one of the two enzymes that catalyze the reactions of the glycerol phosphate shuttle. The activity of the other enzyme of the glycerol phosphate shuttle, mitochondrial glycerol phosphate dehydrogenase (EC 1.1.99.5) (mGPD), is abundant in tissues, such as brain, skeletal muscle and the pancreatic islet, suggesting that the glycerol phosphate shuttle is important in these tissues which rapidly metabolize glucose. Cytosolic malic enzyme activity is important for shuttles which transport NADPH equivalents from mitochondria to the cytosol. The major finding of the study was a highly abnormal metabolite pattern in tissues of the mmgg mouse suggesting a block in the glycerol phosphate shuttle due to cGPD deficiency. The metabolite pattern did not suggest that malic enzyme deficiency caused an abnormality. Tissue levels of glycerol phosphate (low) and dihydroxyacetone phosphate (high) were only abnormal in skeletal muscle. Glycolytic intermediates, situated at or before the triose phosphates in the pathway, such as fructose bisphosphate and glyceraldehyde phosphate were increased depending on the tissue. Taken together with previous extensive data on the mouse deficient only in cGPD this suggests a block in glycolysis at the step catalyzed by glyceraldehyde phosphate dehydrogenase caused by an abnormally low NAD/NADH ratio resulting from a nonfunctional glycerol phosphate shuttle. Consistent with this idea the lactate/pyruvate ratio was high in skeletal muscle signifying a low cytosolic NAD/NADH ratio. The mmgg mouse was normal in all other factors studied including blood glucose and serum insulin levels, pancreatic islet mass, insulin release from isolated pancreatic islets, as well as the activities of five metabolic enzymes, including mGPD, in liver, kidney, skeletal muscle and pancreatic islets. cGPD enzyme activity was undetectable in pancreatic islets, 0.5% of normal in liver, and 2.1% of normal in kidney and skeletal muscle. Malic enzyme activity was undetectable in these same tissues.     

Activation of fructose 1,6-bisphosphatase in darkened intact chloroplasts by NADPH

When intact chloroplasts are incubated in the dark with dihydroxyacetone phosphate, an increase in fructose 1,6-bisphosphatase activity occurs which resembles the reductive activation observed in illuminated chloroplasts. Under optimum conditions, the activity increases to about 150 μmol · h^?1 · mg^?1 chlorophyll within 60 min. The dark activation of the enzyme is reversed by electron acceptors such as oxaloacetate, nitrite, and 3-phosphoglycerate plus ATP. Activation is most marked under strictly anaerobic conditions, being strongly inhibited by O₂. It is concluded that NADPH, generated from dihydroxyacetone phosphate in situ in the reaction catalyzed by NADP⁺-dependent glyceraldehyde phosphate dehydrogenase, can provide electrons for the reductive activation of fructose 1,6-bisphosphatase in the dark.     

Regulation of NADP-malate dehydrogenase in C4 plants: relationship among enzyme activity, NADPH to NADP ratios, and thioredoxin redox states in intact maize mesophyll chloroplasts

NADP-malate dehydrogenase activity, the ratio of NADPH to NADP, and thioredoxin redox state in Zea mays chloroplasts were determined after various treatments. Following transfer from dark to light, NADP-malate dehydrogenase was activated more than 20-fold within 10 min while the proportion of pyridine nucleotide as NADPH increased from about 25 to 90%, and the proportion of thioredoxin in the reduced form increased from 20 to more than 90%, in less than 1 min. After transfer back to the dark, NADPH levels dropped very rapidly to the initial values recorded before illumination, while enzyme activity and reduced thioredoxin levels decreased more slowly. Addition of oxaloacetate or 3-phosphoglycerate to illuminated chloroplasts results in a decrease of about 70% in the activity of NADP-malate dehydrogenase, a 30% decrease in the level of NADPH, and a 25% decrease in the reduced thioredoxin content. Adding dihydroxyacetone phosphate and pyruvate had no effect. These results are considered in relation to the hypothesis that NADP-malate dehydrogenase activity in chloroplasts may be determined by factors regulating the ratio of NADPH to NADP as well as those influencing the redox state of thioredoxin.     

The tritium isotope effect of sn-glycerol 3-phosphate oxidase and the effects of clofenapate and N-(2-benzoyloxyethyl)norfenfluramine on the esterification of glycerol phosphate and dihydroxyacetone phosphate by rat liver mitochondria

1. Owing to a (3)H isotope effect, the mitochondrial sn-glycerol 3-phosphate oxidase (EC 1.1.99.5) had a mean activity which was 8.4 times less with sn-[2-(3)H]-rather than with sn-[1-(14)C]glycerol 3-phosphate as a substrate. 2. A method for measuring the simultaneous synthesis of lipid from glycerol phosphate and dihydroxyacetone phosphate in rat liver mitochondria is described. 3. The lipid synthesized by rat liver mitochondria from sn-[1-(14)C]glycerol 3-phosphate was mainly phosphatidate and lysophosphatidate, whereas that synthesized from dihydroxy[1-(14)C]acetone phosphate was mainly acyldihydroxyacetone phosphate. 4. Additions of NADPH facilitated the conversion of acyldihydroxyacetone phosphate into lysophosphatidate and phosphatidate. 5. Hydrazine (1.4mm) or KCN (1.4mm) inhibited the synthesis of lipids from dihydroxyacetone phosphate but not from glycerol phosphate. 6. Clofenapate (1-2.5mm) inhibited the synthesis of lipids from dihydroxyacetone phosphate but slightly stimulated synthesis from glycerol phosphate. 7. The methanesulphonate of N-(2-benzoyloxyethyl)norfenfluramine, at 0.25-0.75mm, inhibited lipid synthesis from both glycerol phosphate and dihydroxyacetone phosphate.     

A functional arginine residue in NADPH-dependent aldehyde reductase from pig kidney.

Pig kidney aldehyde reductase is inactivated by 2,3-butanedione, phenylglyoxal, methylglyoxal, and 1,2-cyclohexanedione. 2,3-Butanedione caused the most rapid loss in enzyme activity, the rate of loss being proportional to the concentration of 2,3-butanedione. Neither D-glyceraldehyde nor pyridine 3-aldehyde, both substrates for this broadly specific enzyme, protected the enzyme from inactivation but 1 mM NADPH or NADP completely prevented the loss of activity by 2,3-butanedione suggesting the involvement of arginine in the binding of cofactor. Nicotinamide mononucleotide (NMN) (reduced form) offered no protection to inactivation whereas ADP-ribose phosphate gave complete protection indicating that it is the latter portion of NADPH which interacts with the essential arginine. Both NMN and ADP-ribose phosphate are competitive inhibitors of aldehyde reductase with respect to NADPH. Butanedione-modified aldehyde reductase could still bind to a blue dextran-Sepharose 4B column suggesting that the modified arginine did not bind NADPH. This was confirmed by fluorescence spectra which showed that chemically modified aldehyde reductase caused the same blue shift of NADPH fluorescence as did native aldehyde reductase. Of additional interest was the quenching of NADPH fluorescence by aldehyde reductase which, with one exception, is in contrast to the fluorescence behavior of all other oxidoreductases.     

Metabolic concomitants in pure, pancreatic beta cells during glucose-stimulated insulin secretion

The role of the redox potential in insulin secretion by beta cells stimulated with high glucose was investigated using an in vitro pancreas perfusion system. To assess glycolytic flux the sum of fructose-1,6-P2 + triose-P was determined in pure beta cells microdissected from lyophilized sections of the isolated perfused pancreas quick frozen during the early insulin secretory response. L-Glycerol 3-phosphate and dihydroxyacetone phosphate were measured as indicators of the free cytosolic [NAD+]/[NADH] ratio and NADH and NADPH were also measured. Fructose-1,6-P2 + triose-P was increased in beta cells simultaneously with the onset of insulin secretion indicating an increase in glucose metabolism had occurred. The ratio of [dihydroxyacetone phosphate]/[L-glycerol 3-phosphate] increased simultaneously with the onset of insulin secretion. NADH content increased only after initiation of insulin secretion and NADPH levels remained unchanged during the early secretory response to high glucose. These data contradict the hypothesis that insulin secretion is triggered by a more reduced cytosolic redox state and instead indicate that insulin secretion is initiated by other metabolic coupling factor(s) generated in beta cells stimulated by high glucose.     

Peroxisomal dihydroxyacetone phosphate acyltransferase. Effect of acetaldehyde on the intact and solubilized activity

The peroxisomal enzyme dihydroxyacetone phosphate (DHAP) acyltransferase shows a differential response to acetaldehyde. Employing whole peroxisomes, the enzyme displays a 130-400% stimulation of activity when assayed in the presence of 10-250 mM acetaldehyde. Following taurocholate solubilization of the enzyme the response to 0.25 M acetaldehyde is one of almost total inhibition. This inhibition of the taurocholate-solubilized enzyme is not observed at acetaldehyde concentrations below 200 mM. The stimulation of DHAP acyltransferase by acetaldehyde is solely a response of the peroxisomal enzyme as evidenced by its insensitivity to N-ethylmaleimide and 5 mM glycerol 3-phosphate. Furthermore, microsomal dihydroxyacetone phosphate acyltransferase activity is inhibited at all acetaldehyde concentrations. The activation of membrane-bound DHAP acyltransferase by acetaldehyde appears to be specific for this enzyme in comparison to several other peroxisomal and microsomal enzymes. The specificity of activation and differential response of the peroxisomal enzyme to acetaldehyde indicates that the microenvironment of the peroxisomal membrane is important for normal enzymatic function of this enzyme.     

Acyl-CoA:dihydroxyacetone phosphate acyltransferase in human skin fibroblasts: study of its properties using a new assay method

In relation to the finding that human skin fibroblasts are capable of de novo either phospholipid biosynthesis, we have studied the properties of acyl-CoA:dihydroxyacetone phosphate acyltransferase in fibroblast homogenates using a new assay method. The results indicate that the acylation of dihydroxyacetone phosphate shows an optimum at pH 5.5 with a broad shoulder of activity up to pH 6.4 and a decline in activity up to pH 8.2. At pH 5.5 the acyltransferase accepts dihydroxyacetone phosphate, but not glycerol 3-phosphate as a substrate. Furthermore, the transferase activity was found to be membrane-bound and inactivated by Triton X-100 at concentrations above 0.025% (w/v). Similar properties have been described for the enzyme as present in rat-liver and guinea-pig liver peroxisomes. These data, together with the finding that acyl-CoA:dihydroxyacetone phosphate acyltransferase is deficient in cultured skin fibroblasts from patients without peroxisomes (Zellweger syndrome), suggest that in cultured skin fibroblasts the enzyme is primarily located in peroxisomes.     

A simple approach to detect active-site-directed enzyme-enzyme interactions. The aldolase/glycerol-phosphate-dehydrogenase enzyme system

A novel approach has been elaborated to identify the mechanism of intermediate transfer in interacting enzyme systems. The aldolase/glycerol-3-phosphate-dehydrogenase enzyme system was investigated since complex formation between these two enzymes had been demonstrated. The kinetics of dihydroxyacetone phosphate conversion catalyzed by the dehydrogenase in the absence and presence of aldolase was analyzed. It was found that the second-order rate constant (kcat/Km) of the enzymatic reaction decreases due to the formation of a heterologous complex. The decrease could be attributed to an increase of the Km value since kcat did not change in the presence of aldolase. In contrast, an apparent increase in the second-order rate constant of dihydroxyacetone phosphate conversion by the dehydrogenase was observed if the triose phosphate was produced by aldolase from fructose 1,6-bisphosphate (consecutive reaction). Moreover, no effect of dihydroxyacetone phosphate on the dissociation constant of the heterologous enzyme complex could be detected by physico-chemical methods. The results suggest that the endogenous dihydroxyacetone phosphate produced by aldolase complexed with dehydrogenase is more accessible for the dehydrogenase than the exogenous one, the binding of which is impeded due to steric hindrance by bound aldolase.     

Purification and properties of an NADPH-dependent dihydroxyacetone reductase from Phycomyces spores

Abstract A glycerol:NADP⁺ 2-oxidoreductase was purified to homogeneity from Phycomyces blakesleeanus sporangiospores. The enzyme had an M _r of 34 000–39 000 and consisted of a single polypeptide. It had a pH optimum between 6–6.5 and a K _m of 3.9 mM for dihydroxyacetone. The reverse reaction had a pH optimum of 9.4 and a K _m for glycerol of more than 2 M. The enzyme was completely specific for NADPH ( K _m= 0.01 mM) or NADP⁺ ( K _m= 0.17 mM) and greatly preferred dihydroxyacetone over glyceraldehyde as substrate. Besides glycerol, l -arabitol and mesoerythritol were also oxidized by the enzyme. It was inhibited by ionic strengths in excess of 100 mM and is probably involved in the synthesis of glycerol during early spore germination.