Molecule Of NADPH Research Articles

A glutamine 67--> histidine mutation in homotetrameric R67 dihydrofolate reductase results in four mutations per single active site pore and causes substantial substrate and cofactor inhibition.

R67 dihydrofolate reductase (DHFR) is a type II DHFR produced by bacteria as a resistance mechanism to increasing clinical use of the antibacterial drug trimethoprim. Type II DHFRs are not homologous in either sequence or structure with chromosomal DHFRs. The crystal structure of R67 DHFR shows a single active site pore that spans the length of the homotetramer. Related sites (due to a 222 symmetry element at the center of the pore) are used to bind ligands, i.e. each half of the pore can accommodate either the substrate, dihydrofolate (DHF), or the cofactor, NADPH, although DHF and NADPH are bound differently. To evaluate the role of glutamine 67 (and its symmetry-related Q167, Q267 and Q367 residues which occur at the center of the active site pore), a Q67H mutation was constructed. Binary binding of dihydrofolate (DHF; monitored by isothermal titration calorimetry) displays two identical sites with a Kd value of 0.04 microM, while binding of NADPH shows two sites possessing negative cooperativity with Kd values of 0.027 and 0.62 microM. A comparison of ligand binding in Q67H versus wild-type (wt) R67 DHFR indicates both ligands bind more tightly (80-6000-fold) and DHF binding in Q67H R67 DHFR no longer displays positive cooperativity as seen in wt R67 DHFR. Ternary complex binding in the Q67H mutant indicates a total of two ligands can bind per pore. Substantial substrate and cofactor inhibition are observed during catalysis, consistent with non-productive binding of either two DHF or two NADPH molecules in Q67H R67 DHFR. Because of the symmetry-related binding sites in the active site pore, the accumulation of potentially positive mutations in R67 DHFR is limited by the balance between tighter binding of ligands (and thus potentially increased catalytic efficiency) and inhibition that arises upon tighter binding of two identical ligands at symmetry-related sites.

The mechanism of thioredoxin reductase from human placenta is similar to the mechanisms of lipoamide dehydrogenase and glutathione reductase and is distinct from the mechanism of thioredoxin reductase from Escherichia coli.

Thioredoxin reductase, lipoamide dehydrogenase, and glutathione reductase are members of the pyridine nucleotide-disulfide oxidoreductase family of dimeric flavoenzymes. The mechanisms and structures of lipoamide dehydrogenase and glutathione reductase are alike irrespective of the source (subunit M(r) approximately 55,000). Although the mechanism and structure of thioredoxin reductase from Escherichia coli are distinct (M(r) approximately 35,000), this enzyme must be placed in the same family because there are significant amino acid sequence similarities with the other two enzymes, the presence of a redox-active disulfide, and the substrate specificities. Thioredoxin reductase from higher eukaryotes on the other hand has a M(r) of approximately 55,000 [Luthman, M. & Holmgren, A. (1982) Biochemistry 21, 6628-6633; Gasdaska, P. Y., Gasdaska, J. R., Cochran, S. & Powis, G. (1995) FEBS Lett 373, 5-9; Gladyshev, V. N., Jeang, K. T. & Stadtman, T.C. (1996) Proc. Natl. Acad. Sci. USA 93, 6146-6151]. Thus, the evolution of this family is highly unusual. The mechanism of thioredoxin reductase from higher eukaryotes is not known. As reported here, thioredoxin reductase from human placenta reacts with only a single molecule of NADPH, which leads to a stable intermediate similar to that observed in titrations of lipoamide dehydrogenase or glutathione reductase. Titration of thioredoxin reductase from human placenta with dithionite takes place in two spectral phases: formation of a thiolate-flavin charge transfer complex followed by reduction of the flavin, just as with lipoamide dehydrogenase or glutathione reductase. The first phase requires more than one equivalent of dithionite. This suggests that the penultimate selenocysteine [Tamura, T. & Stadtman, T.C. (1996) Proc. Natl. Acad. Sci. USA 93, 1006-1011] is in redox communication with the active site disulfide/dithiol. Nitrosoureas of the carmustine type inhibit only the NADPH reduced form of human thioredoxin reductase. These compounds are widely used as cytostatic agents, so this enzyme should be studied as a target in cancer chemotherapy. In conclusion, three lines of evidence indicate that the mechanism of human thioredoxin reductase is like the mechanisms of lipoamide dehydrogenase and glutathione reductase and differs fundamentally from the mechanism of E. coli thioredoxin reductase.

Oxygenic Photoautotrophic Growth and Photosystem I

J. W. Lee et al . ([1][1]) state that the minimum quantum requirement for photosystem II (PSII) photosynthesis should be four photons per O2 molecule, because PSII photosynthesis uses a single light reaction (PSII) instead of two (both PSI and PSII). This statement is not correct because photosynthesis requires adenosine triphosphate (ATP) in addition to reducing power such as that provided by nicotinamide adenine dinucleotide phosphate (NADP+ → NADPH). If one assumes textbook values, the reduction of one CO2 molecule by the enzymes of the Calvin cycle requires 2NADPH and 3ATP. The reduction of two molecules of nicotinamide adenine dinucleotide phosphate (2NADP+) by 2H2O requires only four photons in PSII photosynthesis, but the formation of 3ATP requires another two-and-a-half photons. Here is the calculation: 3ATP requires nine protons to pass through ATP synthase. Four of those protons are provided by the splitting of H2O: ![Graphic][2] The other five protons are assumed to be provided by cyclic electron flow through the cytochrome b/f complex. The requirement is one electron (one photon) for every two protons pumped by the cytochrome b/f complex. Thus, an additional two-and-a-half photons are required for the synthesis of ATP and a total of six-and-a-half photons is the theoretical minimum required to drive PSII CO2 fixation by the Calvin cycle. 1. 1.[↵][3] 1. J. W. Lee, 2. C. V. Tevault, 3. T. G. Owens, 4. E. Greenbaum , Science 273, 364 (1996). [OpenUrl][4][Abstract][5] # Response {#article-title-2} Olson correctly points out that fixation of a single molecule of CO2 in photosynthesis requires a minimum expenditure of three molecules of ATP and two molecules of NADP. He implicitly assumes that the four electrons required to produce the two NADPH molecules are derived from water oxidation through photochemistry in the PSII reaction center. This same reaction releases four protons into the thylakoid lumen. Production of 3ATP requires a total of nine protons, thus five additional protons are required. It is on the production of these five “extra” protons that the discussion of quantum requirement hinges. Olson explicitly assumes that the five protons are derived from cyclic electron flow through the cytochrome b/f complex, a process that pumps two protons into the lumen for each electron that is passed through the complex (and thus the need for two-and-a-half additional photons). The driving reaction for this cyclic flow was not specified, but must be assumed to be PSII because our mutants lack detectable PSI. There is an additional assumption in Olson's calculation—that the linear flow from water to NADP+ is not coupled to any additional proton translocation. Photosynthesis in the absence of PSI would require that one define a pathway for reduction of ferredoxin and ultimately NADP+ by electrons derived from PSII. Recent inhibitor studies by Lee and Greenbaum. ([1][6]) have suggested that the plastoquinone pool and the cytochrome b/f complex may be required in this pathway, but this does not make an understanding of the pathway any clearer. Nevertheless, there is increasing data to support the original observation that some mutants of Chlamydomonas reinhardtii lacking PSI exhibit light-dependent CO2 fixation, or H2 evolution with simultaneous O2 evolution, or both. If the cytochrome b/f complex does participate in direct electron flow to ferredoxin, the necessity for the additional two-and-a-half photons may be obviated and a theoretical minimum quantum requirement of four may be approached. Consideration of quantum requirement is secondary to the problem of establishing the pathway of electrons from PSII to ferredoxin in the absence of PSI. Knowledge of this pathway could then allow one to establish a minimum quantum requirement based on the extent of proton translocation in the pathway. It is on an understanding of this pathway that our efforts should be focused, for herein lies the unique aspects of oxygenic photosynthesis in the absence of PSI. 1. 1.[↵][7] 1. J. W. Lee, 2. E. Greenbaum , unpublished observations. [1]: #ref-1 [2]: /embed/inline-graphic-1.gif [3]: #xref-ref-1-1 View reference 1. in text [4]: {openurl}?query=rft.jtitle%253DScience%26rft.stitle%253DScience%26rft.issn%253D0036-8075%26rft.aulast%253DLee%26rft.auinit1%253DJ.%2BW.%26rft.volume%253D273%26rft.issue%253D5273%26rft.spage%253D364%26rft.epage%253D367%26rft.atitle%253DOxygenic%2BPhotoautotrophic%2BGrowth%2BWithout%2BPhotosystem%2BI%26rft_id%253Dinfo%253Adoi%252F10.1126%252Fscience.273.5273.364%26rft_id%253Dinfo%253Apmid%252F8662525%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [5]: /lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6Mzoic2NpIjtzOjU6InJlc2lkIjtzOjEyOiIyNzMvNTI3My8zNjQiO3M6NDoiYXRvbSI7czoyMjoiL3NjaS8yNzUvNTMwMi85OTYuYXRvbSI7fXM6ODoiZnJhZ21lbnQiO3M6MDoiIjt9 [6]: #ref-2 [7]: #xref-ref-2-1 View reference 1. in text

Fluorescence-energy transfer in human estradiol 17 beta-dehydrogenase-NADPH complex and studies on the coenzyme binding,.

Fluorescence spectroscopy was used to examine the interaction between human estradiol 17 beta-dehydrogenase (estrogenic 17 beta-hydroxysteroid dehydrogenase, 17 beta-HSD) and the cofactor NADPH. After the binding of NADPH to the enzyme, there was an emission enhancement at 436 nm following an excitation at 295 nm, as compared to the cofactor alone. This phenomenon was attributed to a radiationless transfer of excitation energy from 17 beta-HSD to the enzyme-bound cofactor. The distance of 2.69 nm, between the bound NADPH and the sole tryptophan residue (Trp46) within one subunit, has been determined using fluorescence energy transfer. This result coincides very well with the same distance, recently calculated from the crystallographic coordinates obtained by Ghosh et al. [Ghosh, D., Pletnev, V. Z., Zhu, D.-W., Wawrzak, Z., Duax, W. L., Pangborn, W., Labrie, F. & Lin, S.-X. (1995) Structure 3, 503-513]. Compared to free NADPH, the fluorescence emission of enzyme-bound NADPH was increased in intensity and its maximum blue-shifted from 457 nm to 436 nm. Binding of NADPH to 17 beta-HSD was studied by fluorescence titration. The enzyme binds two molecules of NADPH with a Kd = 0.73 +/- 0.2 microM. The dissociation constant was further confirmed by the method of coenzyme protection against cold inactivation of the enzyme. The binding was little altered in the presence of estradiol-17 beta. The environment of tryptophan residues on the surface of the enzyme is discussed.

Unusual Binding Stoichiometries and Cooperativity Are Observed during Binary and Ternary Complex Formation in the Single Active Pore of R67 Dihydrofolate Reductase, a D2 Symmetric Protein

R67 dihydrofolate reductase (DHFR) is an R-plasmid-encoded enzyme that confers resistance to the antibacterial drug, trimethoprim. This DHFR variant is not homologous in either sequence or structure to chromosomal DHFRs. A recent crystal structure of the active tetrameric species describes a single active site pore that traverses the length of the protein (Narayana et al., 1995). Related sites (due to a 222 symmetry element at the center of the active site pore) are used for binding of ligands, i.e., each half-pore can accommodate either the substrate, dihydrofolate, or the cofactor, NADPH, although dihydrofolate and NADPH are bound differently. Ligand binding in R67 DHFR was evaluated using time-resolved fluorescence anisotropy and isothermal titration calorimetry techniques. Under binary complex conditions, two molecules of either NADPH, folate, dihydrofolate, or N10 propargyl-5,8-dideazafolate (CB3717) can be bound. Binding of NADPH displays negative cooperativity, binding of either folate or dihydrofolate shows positive cooperativity, and binding of CB3717 shows two identical sites. Any asymmetry introduced by binding of one ligand is proposed to induce the cooperativity associated with binding of the second ligand. Evaluation of ternary complex formation demonstrates that one molecule of folate binds to a 1:1 mixture of R67 DHFR+NADPH. These binding results indicate a maximum of two ligands bind in the pore. A mechanism describing catalysis is proposed that is consistent with the binding results.

Crystal Structure ofEscherichia coliQOR Quinone Oxidoreductase Complexed with NADPH

The crystal structure of the homodimer of quinone oxidoreductase fromEscherichia colihas been determined using the multiple isomorphous replacement method at 2.2 Å resolution and refined to an R-factor of 14.1%. The crystallographic asymmetric unit contains one functional dimer with the two subunits being related by a non-crystallographic 2-fold symmetry axis. The model consists of two polypeptide chains (residues 2 through 327), one NADPH molecule and one sulphate anion per subunit, and 432 water molecules. Each subunit consists of two domains: a catalytic domain and a nucleotide-binding domain with the NADPH co-factor bound in the cleft between domains. Quinone oxidoreductase has an unusual nucleotide-binding fingerprint motif consisting of the sequence AXXGXXG. The overall structure of quinone oxidoreductase shows strong structural homology to that of horse liver alcohol dehydrogenase.f2f2Abbreviations used: QOR, quinone oxidoreductase; LADH, liver alcohol dehydrogenase; GDH, glucose dehydrogenase; MIR, multiple isomorphous replacement; SA, simulated annealing; NCS, non-crystallographic symmetry.

Imaging neutrophil activation: analysis of the translocation and utilization of NAD(P)H-associated autofluorescence during antibody-dependent target oxidation.

Fluorescence intensified/enhanced microscopy has been used to study the metabolic activation of living human neutrophils in time-lapse sequences. The autofluorescence associated with NAD(P)H's emission band was studied within individual quiescent and stimulated cells. Excitation of NAD(P)H-associated autofluorescence was provided by a high-intensity Hg-vapor lamp. The background-subtracted autofluorescence signals were computer enhanced. In some cases the ratio image of NAD(P)H-associated autofluorescence to tetramethyl-rhodamine methyl ester (TRME) fluorescence, which was found to be uniformly distributed within neutrophils, was calculated to normalize autofluorescence intensities for cell thickness. Activation of the NADPH oxidase by phorbol myristate acetate, F-, N-formyl-methionyl-leucyl-phenylalanine (FMLP), or tumor necrosis factor (TNF) dramatically reduced autofluorescence levels. Membrane solubilization with sodium dodecyl sulfate eliminated autofluorescence. Thus, control experiments indicated that most or all of the detectable NAD(P)H-associated autofluorescence was due to NAD(P)H, consistent with previous non-microscopic studies. To understand the metabolic events surrounding the internalization and oxidative destruction of targets, we have imaged the NAD(P)H-associated autofluorescence of neutrophils and the Soret band of antibody coated target erythrocytes during cell-mediated cytotoxicity. Absorption contrast microscopy of the erythrocyte's Soret band is an especially sensitive indicator of the entry of reactive oxygen metabolites into this target's cytosol. Thus, it is possible to spectroscopically dissect and image the substrate (NADPH) and product (O2-) reactions of the NADPH oxidase in living unlabeled neutrophils. During real-time experiments at 37 degrees C, the level of NAD(P)H-associated autofluorescence surrounding phagosomes greatly increases before the disappearance of the target's Soret band. NAD(P)H-associated autofluorescence in the vicinity of phagocytosed erythrocytes is greatly diminished after target oxidation. This suggests that NAD(P)H is translocated to the vicinity of phagosomes prior to the oxidation of targets. The apparent cytosolic redistribution of NAD(P)H was confirmed by ratio imaging microscopy to control for cell thickness. We suggest that NADPH including its sources and/or carriers accumulate near phagosomes prior to target oxidation and that local NADPH molecules are consumed during target oxidation.

A new property of catalase: the concerted synthesis of nucleotide triphosphates

We have observed a new property of catalase: the in vitro concerted synthesis of triphosphate nucleotides (XTP) from the corresponding diphosphate nucleotides (XDP), a reaction which is greatly enhanced in the presence of H 2O 2. Kinetic data and inhibition studies showed that the reaction is neither of the adenylate kinase type, nor of the nucleoside-diphosphate kinase type. Although the reaction depends on the presence of phosphate buffer, inorganic phosphate was not used in the phosphorylation reaction. ATP production did not occur with NADPH-depleted catalase, but was restored when the D-catalase was preincubated in the presence of NADPH and H 2O 2. These data suggest that the NADPH molecule may be the phosphate donor in the reaction. The possible physiological significance of the reaction is discussed.

A new property of catalase: The concerted synthesis of nucleotide triphosphates

We have observed a new property of catalase: the in vitro concerted synthesis of triphosphate nucleotides (XTP) from the corresponding diphosphate nucleotides (XDP), a reaction which is greatly enhanced in the presence of H 2O 2. Kinetic data and inhibition studies showed that the reaction is neither of the adenylate kinase type, nor of the nucleoside-diphosphate kinase type. Although the reaction depends on the presence of phosphate buffer, inorganic phosphate was not used in the phosphorylation reaction. ATP production did not occur with NADPH-depleted catalase, but was restored when the D-catalase was preincubated in the presence of NADPH and H 2O 2. These data suggest that the NADPH molecule may be the phosphate donor in the reaction. The possible physiological significance of the reaction is discussed.

ATP turnover by the fatty acid reductase complex of Photobacterium phosphoreum

A sensitive reverse-phase high pressure liquid chromatographic assay for formation of AMP coupled with analysis of aldehyde production has been used to characterize the properties of the fatty acid reductase complex of Photobacterium phosphoreum. The enzyme complex, which consists of three different polypeptides (34 000, 50 000, and 58 000), has a high affinity for ATP (Km = 20 nM) and shows highest specificity with C14 fatty acids. Activation of the fatty acid is efficiently coupled to the reduction step showing a stoichiometry of one molecule of fatty acid reduced to aldehyde and one molecule of NADPH oxidized for every molecule of ATP converted to AMP. Reconstituted fatty acid reductase (50 000 and 58 000) shows an ATP hydrolase activity that is independent of NADPH with the maximum amount of AMP formed limited by the amount of fatty acid in the assay, consistent with acyl-protein turnover experiments and the channeling of fatty acids to form acyl thioesters (−NADPH) or aldehyde (+NADPH). Addition of the 34 000 polypeptide to the reconstituted enzyme results in stimulation of AMP formation (−NADPH) to a level far exceeding the amount of fatty acid, showing that the fatty acid can be recycled by the 34 000 protein through its thioesterase activity. Also the 34 000 protein is responsible for a two- to three-fold stimulation in the rate of ATP hydrolysis, suggesting that it can be involved in the stabilization of the enzyme complex.

NADPH oxidation catalyzed by the peroxidase/H2O2 system

Oxidation of NADPH catalyzed by the peroxidase/H2O2 system is known to require the presence of mediating molecules. Using either lactoperoxidase or horseradish peroxidase, we demonstrated that in the peroxidase/H2O2 system, NADPH oxidation was mediated by iodide. The oxidation product was the iodinated NADP. This product was shown to possess spectral characteristics different from those of NADP+ and NADPH, since for iodinated NADP, increased absorbance was observed in the 280-nm region and was directly proportional to the rate of iodination. It is suggested that oxidation and iodination of NADPH proceed via a single reaction between the intermediary iodide oxidation species and NADPH. Experiments with different molecules of NADPH analogues indicated that iodination occurred in the nicotinamide part of the NADPH molecule.

Catalase: a tetrameric enzyme with four tightly bound molecules of NADPH.

Catalases (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) from many species are known to be tetramers of 60,000-dalton subunits, with four heme groups per tetramer. Previous authors have determined the amino acid sequence and three-dimensional structure of bovine liver catalase. Studies of the regulation of the pentose phosphate pathway led the present authors to a search for proteins that bind NADP+ and NADPH in human erythrocytes. An unexpected result of that search was the finding that a major reservoir of bound NADPH in human erythrocytes is catalase. Each tetrameric molecule of human or bovine catalase contains four molecules of tightly bound NADPH. The binding sites have the relative affinities NADPH greater than NADH greater than NADP+ greater than NAD+. NADPH does not seem to be essential for the enzymic conversion of H2O2 to O2 and water but does provide protection of catalase against inactivation by H2O2.

Mechanism of Glyoxylate Decarboxylation in the Glycolate Pathway in Euglena gracilis Z

The mechanism for decarboxylating the carboxyl carbon of glycolate was studied in Euglena gracilis Z, which liberates more than 70% of the carboxyl carbon as CO(2) during glycolate metabolism.In the isolated mitochondria of E. gracilis, glycolate was oxidized to glyoxylate, 25% of which was aminated to glycine, with the remaining unchanged in the presence of glutamate; in the absence of the amino donor, glyoxylate was not changed. Irrespective of the presence or absence of the amino donor, no decarboxylation took place in Euglena mitochondria. In Euglena chloroplasts glyoxylate was actively decarboxylated by the action of hydrogen peroxide generated by the reaction of a Mn(2+)-dependent NADPH oxidase. This enzyme was purified 120-fold over the crude extract and some of its properties studied. Oxidation of one molecule of NADPH was accompanied by the formation of one molecule of H(2)O(2); the NADPH oxidation was not attributable to the action of l-ascorbate peroxidase, the sole peroxidase present in E. gracilis. Specific participation of chloroplasts and mitochondria in glycolate metabolism is discussed.

Alcohol dehydrogenase from leuconostoc mesenteroides: molecular properties in comparison with the yeast and horse liver enzymE

1. 1. Leuconostoc mesenteroides alcohol dehydrogenase is an acidic protein (pI =4.1) with a mol wt of 135,000 consisting of 4 identical subunits. 2. 2. It contains 2 Zn atoms and 5 cysteine residues per subunit. 3. 3. Thermal stability increases with increasing ionic strength. 4. 4. One NADPH molecule is bound per subunit. NADPH is bound about 10 times more strongly than NADH. Fluorescence spectra of bound coenzymes show characteristic blue shifts and intensity enhancements compared to those of free NAD(P)H. 5. 5. A comparison of molecular properties between horse liver-, yeast- and L. mesenteroides alcohol dehydrogenase shows the closer relationship of Leuconostoc ADH to yeast ADH.

PH-Dependent thermodynamic parameters of the glutamate dehydrogenase-α-ketoglutarate-NADPH complex

The enthalpy of formation of the reactive bovine glutamate dehydrogenase-α-ketoglutarate-NADPH complex has been measured as a function of pH, at two temperatures and in two buffer systems having different enthalpies of ionization. The results demonstrate the existence of an extensive two-way traffic of protons between the buffer and the complex itself. While the pattern in a single buffer is too complex to resolve within the experimental limitations imposed by the system, we develop a theory which shows that the difference in ΔH° H + between buffers of different enthalpies of ionization has a simpler, more easily resolvable form. From such a relationship we show that in the process of binding of a molecule of NADPH to the enzyme-α-ketoglutarate complex, a proton has been shifted from the buffer to the complex and that this is due to the shift of the pK of an ionized group on the enzyme to a much higher value when the complex is formed. The ΔC p of formation of the complex is −450 cal · deg. −1 · M −1, and is pH independent. Implications of these results for the interpretation of enthalpies of enzyme-complex formation obtained at a single pH, and at one temperature, with one buffer system are mentioned. The complexity of the pattern of the dependence of ΔH°, taken together with the magnitudes of the individual features of these dependencies, suggest that a number of large pK shifts occur when the enzyme-NADPH-α-keto-glutarate complex is formed. It is quite probable that these shifts represent a sizeable fraction of the machinery of the catalytic mechanism.

Kinetic analysis of the reaction catalyzed by rat-liver 3-hydroxy-3-methylglutaryl-coenzyme-A reductase using two specific inhibitors.

The mechanism of action of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase solubilized from rat liver microsomes has been investigated. In the reduction of 3‐hydroxy‐3‐methylglutaryl‐CoA to mevalonate, an overall initial velocity study gave a linear intersecting pattern when both 3‐hydroxy‐3‐methylglutaryl‐CoA and NADPH were variable substrates. Adenosine 2′‐monophospho‐5′‐diphos‐phoribose, which was found to be a reversible inhibitor of reductase, inhibited the enzyme competitively with respect to NADPH, and uncompetitively with respect to 3‐hydroxy‐3‐methylglutaryl‐CoA. On the other hand, the inhibition of reductase by ML‐236B (sodium salt), a specific reversible inhibitor of the enzyme isolated from the culture of a Penicillium (whose structure is given in the paper) is competitive with respect to 3‐hydroxy‐3‐methylglutaryl‐CoA and noncompetitive with respect to NADPH [Endo et al. (1976) FEBS Lett. 72, 323–3261. The reduction of 3‐hydroxy‐3‐methylglutaryl‐CoA to mevalonate was subject to the substrate inhibition by NADPH attributed to the formation of a productive enzyme‐NADPH complex. These results indicate that the two substrates bind to the enzyme effectively in an ordered manner; reductase first interacts with 3‐hydroxy‐3‐methylglutaryl‐CoA to make a binary complex, which, in turn, forms a ternary complex with one molecule of NADPH. Considered together with the results of product inhibition study, and assuming a hemithioacetal of mevaldate and CoA is an intermediate of the reductase reaction, a bi‐uni‐uni‐ter‐ping‐pong mechanism is proposed as a model of the overall reaction.

Dihydrofolate reductase from Lactobacillus caSci. Stereochemistry of NADPH binding.

The NADPH molecule binds to dihydrofolate reductase in an extended conformation. Several of the individual dihedral angles, especially in the adenine mononucleotide portion of the coenzyme, differ from their minimum energy conformations. The ribose phosphate portions of the coenzyme are involved in numerous specific hydrogen-bonded and charge-charge interactions. The adenine ring resides in an apparently nonspecific hydrophobic cleft and the nicotinamide ring is bound within an intricately constructed cavity, one wall of which includes the pyrazine ring of bound methotrexate. Two rather extended loops (residues 10 to 24 and 117 to 135) connecting beta A to alpha B and beta F to beta G, respectively, move 2 to 3 A when NADPH binds to dihydrofolate reductase. No overall structural homology is evident between the dinucleotide binding domains of dihydrofolate reductase on the one hand and the four NAD+-dependent dehydrogenases of known structure on the other. However, binding does occur in both cases at the carboxyl edge of a region of parallel beta sheet flanked by a pair of alpha helices.

Squalene synthetase.

In the first part of the review the background to the discovery of the asymmetric synthesis of squalene from two molecules of farnesyl pyrophosphate and NADPH is described, then the stereochemistry of the overall reaction is summarized. The complexity of the biosynthesis of squalene by microsomal squalene synthetase demanded the existence of some intermediate(s) between farnesyl pyrophosphate and squalene. This demand was satisfied by the discovery of presqualene pyrophosphate, an optically active C30 substituted cyclopropylcarbinyl pyrophosphate, the absolute configuration of which at all three asymmetric centers of the cyclopropane ring was deduced to be R. Possible mechanisms for the biosynthesis of presqualene pyrophosphate and its reductive transformation into squalene are presented. In the second part of the review the nature of the enzyme is discussed. The question whether presqualene pyrophosphate is an obligate intermediate in the biosynthesis of squalene is examined, with the firm conclusion that it is. It is as yet uncertain whether the two half reactions of squalene synthesis, i.e. (i) 2 x farnesyl pyrophosphate leads to presqualene pyrophosphate; (ii) presqualene pyrophosphate + NADPH (NADH) leads to squalene, are catalyzed by one or two enzymes or by a large complex with two catalytic sites. Evidence is cited for the existence on the enzyme of two distinct binding sites with different affinities for the two farnesyl pyrophosphate molecules. The types of enzyme preparations available at present are described and types of experiments carried out with these are critically examined. The implications of the properties of a low molecular weight squalene synthetase solubilized with deoxycholate from microsomal membranes is discussed and a model for the enzyme in an organized membrane structure is presented.

Mechanism of action of a wound-induced ω-hydroxyfatty acid:NADP oxidoreductase isolated from potato tubers ( Solanum tuberosum L)

ω-Hydroxyfatty acid:NADP oxidoreductase, an enzyme involved in suberin biosynthesis, is induced by wounding potato tubers. Initial velocity and product inhibition studies with the purified enzyme suggested an ordered sequential mechanism, where NADPH is added first, followed by 16-oxohexadecanoate, and NADP is released after 16-hydroxyhexadecanoate. Substrate inhibition by NADPH was observed at concentrations higher than 0.2 m m. The inhibitory NADPH molecule competes with 16-oxohexadecanoate, indicating that it forms a dead-end complex with the E-NADPH form of the enzyme. The kinetics for the NADPH inhibition suggested that n > 1 in the rate equation v = V[NADPH] (K m + [NADPH]+ [NADPH] n+1 K i ) ; i.e., more than two NADPH molecules bind to enzyme. The K m for 16-oxohexadecanoate did not change from pH 7.5 to 9.0 but increased about 10-fold from pH 9.0 to 10.0, whereas the K m for NADPH and hexadecanal did not vary significantly in this pH range. Phenylglyoxal inactivated the enzyme; NADPH and AMP (which competes with NADPH; K i = 1.1 mM) provided protection against such inactivation. Diethylpyrocarbonate also caused inactivation which was reversed by hydroxylamine; NADPH but not AMP protected the enzyme from this inhibition. Pyridoxal-5′-phosphate reversibly inactivated the enzyme and NaBH 4 reduction of the pyridoxal phosphate-treated enzyme resulted in irreversible inhibition; a combination of NADPH and ω-oxo C 16 acid provided protection against such inactivation. As the chain length of alkanals increased from C 3 to C 8, the K m for the substrate decreased drastically from 7000 to 90μ m and a further increase in chain length from C 8 to C 20 resulted in only a small decrease in K m . The K m and V for 8-oxooctanoate and 10-oxodecanoate are compared with the values obtained for 16-oxohexadecanoate. Based on these results, it is proposed that arginine acts as the binding site for NADPH, a hydrophobic crevice with lysine at the bottom forms the binding site for 16-oxohexadecanoate and histidine participates in the reaction as the proton donor.

Purification and properties of NADPH-dependent aldehyde reductase from human liver.

An aldehyde reductase (EC 1.1.1.2) from human liver has been purified to homogeneity. The enzyme is NADPH-dependent, prefers aromatic to aliphatic aldehydes as substrates, and is inhibited by barbiturates and hydantoins. The following physicochemical parameters were determined: molecular weight, 36,200; sedimentation coefficient, 2.9 S; Stokes radius, 2.65 nm; isoelectric point, pH 5.3; extinction coefficient at 280 nm, 54,300 M-1 cm-1. Results from polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, gel filtration, and ultracentrifugation suggest a monomeric structure. On molecule of NADPH binds to the enzyme causing a red shift of the coenzyme absorption maximum from 340 to 352 nm. The amino acid composition has been determined and a partial specific volume of 0.74 was computed from these data. An alpha-helicity of 7 and 18% was estimated from the ellipticities at 208 and 222 nm, respectively. Combination of the most reactive thiol group with p-mercuribenzoate does not cause loss of catalytic activity. Inactivation occurs when more than one thiol group is modified. The presence of NADPH or NADP+ prevents loss of activity by thiol modification. The comparison of structural features of aldehyde reductase with other monomeric and oligomeric dehydrogenases suggest similarities of aldehyde reductase with octopine dehydrogenase.