2'',3''-CYCLIC NADP AS A SUBSTRATE FOR 2'',3''-CYCLIC NUCLEOTIDE 3''-PHOSPHOHYDROLASE

Abstract– 2',3'-Cyclic NADP has been prepared by cyclization of NADP at pH 6 in the presence of l-ethyl-(3-dimethylaminopropyl)-carbodiimide. The NADP derivative is readily hydrolyzed to NADP by the enzyme in brain and nerve that hydrolyzes 2',3'-cyclic nucleotides to 2'-phospho esters. The K _m for this substrate is the same as that for 2',3'-cyclic AMP (0.22 m m ) at pH 6 and 25°C. The two substrates are hydrolyzed by the phosphohydrolase at similar maximum velocities. The nicotinamide moiety in cyclic NADP thus has little effect on the enzyme-substrate interaction. This synthetic substrate can be used in a rapid (2 min) and sensitive (10 ng of 31-fold purified enzyme) spectrophotometric coupled enzyme assay for 2',3'-cyclic nucleotide 3'-phosphohydrolase; in this assay the hydrolysis proceeds in the presence of glucose-6-phosphate dehydrogenase and its substrate and the NADPH formed is measured by the increase in absorbance at 340 nm. The assay is applicable to tissue extracts as well as to purified preparations of the enzyme. There is no interference from nucleases of the pancreatic RNase A type.     

Bioluminescent assay of NADPH-dependent isocitrate dehydrogenase and its substrates and cofactors

A bioluminescent assay for NADPH-dependent isocitrate dehydrogenase and for its substrates and cofactors was developed. The method is based on continuous NADPH monitoring in the reaction. The linear range of the assay for the enzyme activity is from 0.05 U/liter to 30 U/liter. It is about 300 times more sensitive than the corresponding spectrophotometric assay at 340 nm. Good correlation exists between both assays. Isocitrate, NADP, manganese, and magnesium can be measured at picomole levels. The applicability of the assays to serum analysis is discussed.     

A stopped-flow study of the reaction between mercuric reductase and NADPH

The reaction of the FAD-containing enzyme, mercuric reductase, with NADPH has been studied by stopped-flow kinetic methods at 25 degrees C, pH 7.3. The results suggest that the reaction involves at least three steps. The first step is very rapid and is essentially complete within the dead time of the stopped-flow apparatus. This step is associated with decreasing absorbances at 340 nm (NADPH) and 455 nm (FAD), whereas there is little formation of the absorbance at 530 nm characterizing 2-electron-reduced enzyme subunits (EH2). The second step involves an increase of the absorbance at 530 nm. The third step results in an increase of the intensity of the long-wavelength band and a change of its shape. A second equivalent of NADPH per FAD is required for this step. It is proposed that the product is an EH2-NADPH complex. In addition to these rapid steps, slow absorbance changes are also observed.     

Spectroscopic and kinetic characterization of nonenzymic and aldose reductase mediated covalent NADP-glycolaldehyde adduct formation

Reaction of glycolaldehyde with the binary E-NADP complex of bovine kidney aldose reductase (ALR2) produces an enzyme-bound chromophore whose absorbance (lambd max 341 nm) and fluorescence (lambda ex max 341 nm; lambda emit max 421 nm) properties are distinct from those of NADPH or E.NADPH yet are consistent with the proposed covalent adduct structure [1,4-dihydro-4-(1-hydroxy-2-oxoethyl)nicotinamide adenine dinucleotide phosphate]. The kinetics of adduct formation, both in solution and at the enzyme active site, support a mechanism involving rate-determining enolization of glycolaldehyde at high [NADP+] or [E.NADP]. At low [NADP+] or [E.NADP] the reaction is second-order overall, but the ALR2-mediated reaction displays saturation by glycolaldehyde due to competition of the aldehyde (plus hydrate) and enol for E.NADP. Measurement of the pre-steady-state burst of E-adduct formation confirms that glycolaldehyde enol is the reactive species and gives a value of 1.3 x 10(-6) for Kenol = [enol]/[( aldehyde] + [hydrate]), similar to that determined by trapping the enol with I3-. At the ALR2 active site, the rate of adduct formation is enhanced 79,000-fold and the adduct is stabilized greater than or equal to 13,000-fold relative to the reaction with NADP+ in solution. A portion of this enhancement is ascribed to specific interaction of NADP+ with the enzyme since the 3-acetylpyridine analogue, (AP)ADP+, gives values that are 15-200-fold lower. Additional evidence for strong interaction of ALR2 with both NADP+ and NADPH is reported. Yet, because dissociation of adduct is slow, catalysis of the overall adduct formation reaction by ALR2 is less than or equal to 67-fold.     

Simultaneous analysis of NAD- and NADP-linked activities of dual nucleotide-specific dehydrogenases. Application to Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase.

A method is described which enables one to assay simultaneously the NAD- and NADP-linked reactions of dehydrogenases which can utilize both coenzymes. The method is based on the fact that the thionicotinamide analogs of NADH and NADPH absorb light maximally at 400 nm, a wavelength sufficiently far removed from the absorbance maximum of NADH and NADPH to permit measurements of the simultaneous reduction of NAD+ (or NADP+) and the thionicotinamide analog of NADP+ (or NAD+). Application of the method to glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides reveals differential effects of glucose 6-phosphate concentration on the NAD- and NADP-linked reactions catalyzed by this enzyme which can not be detected by conventional assay procedures and which may have regulatory significance.     

Lysine residues 162 and 340 are involved in the catalysis and coenzyme binding of NADP(+)-dependent malic enzyme from pigeon

Alanine-scanning site-directed mutagenesis was carried out on all conserved lysine residues of pigeon cytosolic NADP(+)-dependent malic enzyme. Only two mutant enzymes, K162A and K340A, showed significant effect on their kinetic parameters. Both mutant enzymes have K(m) values for Mn(2+) and l-malate similar to those of wild-type. The K(m) value for NADP(+) of K162A is identical to that of wild-type. However, K162A demonstrated a 235-fold decrease in the k(cat) value (0.17 +/- 0.01 vs 40.0 +/- 1.3 s(-1)). These data suggested that the side chain of K162 is important for the enzyme catalytic reaction. We propose that the epsilon-amino group of K162 may serve as a general acid to protonate the 3-carbon of enolpyruvate after decarboxylation. The K340A mutant demonstrated no effect on the k(cat) value. However, its K(m) value for NADP(+) was increased by a factor of 65 (225.7 +/- 5.07 vs 3.49 +/- 0.05 microM). We propose that the NADP(+) specificity is determined by the electrostatic interaction between the epsilon-amino group of K340 and 2'-phosphate of NADP(+).     

Re-engineering the discrimination between the oxidized coenzymes NAD+ and NADP+ in clostridial glutamate dehydrogenase and a thorough reappraisal of the coenzyme specificity of the wild-type enzyme

Clostridial glutamate dehydrogenase mutants, designed to accommodate the 2'-phosphate of disfavoured NADPH, showed the expected large specificity shifts with NAD(P)H. Puzzlingly, similar assays with oxidized cofactors initially revealed little improvement with NADP(+) , although rates with NAD(+) were markedly diminished. This article reveals that the enzyme's discrimination in favour of NAD(+) and against NADP(+) had been greatly underestimated and has indeed been abated by a factor of >?16,000 by the mutagenesis. Initially, stopped-flow studies of the wild-type enzyme showed a burst increase of A(340) with NADP(+) but not NAD(+), with amplitude depending on the concentration of the coenzyme, rather than enzyme. Amplitude also varied with the commercial source of the NADP(+). FPLC, HPLC and mass spectrometry identified NAD(+) contamination ranging from 0.04 to 0.37% in different commercial samples. It is now clear that apparent rates of NADP(+) utilization mainly reflected the reduction of contaminating NAD(+), creating an entirely false view of the initial coenzyme specificity and also of the effects of mutagenesis. Purification of the NADP(+) eliminated the burst. With freshly purified NADP(+), the NAD(+) : NADP(+) activity ratio under standard conditions, previously estimated as 300 : 1, is 11,000. The catalytic efficiency ratio is even higher at 80,000. Retested with pure cofactor, mutants showed marked specificity shifts in the expected direction, for example, 16 200 fold change in catalytic efficiency ratio for the mutant F238S/P262S, confirming that the key structural determinants of specificity have been successfully identified. Of wider significance, these results underline that, without purification, even the best commercial coenzyme preparations are inadequate for such studies.     

Simplified spectrophotometric assay for microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of coenzyme A

A new assay for 3-hydroxy-3-methylglutaryl CoA reductase (mevalonate:NADP oxidoreductase [acylating CoA], EC 1.1.1.34) is based upon the measurement of released coenzyme A (SH) during the reduction of 3-hydroxy-3-methylglutaryl CoA to mevalonate. Coenzyme A was measured in the presence of dithiothreitol, required for activity, by reaction with 5,5'-dithiobis(2-nitrobenzoic acid). Sodium arsenite forms a complex with the dithiol, but not with monothiols. Thus, reduced coenzyme A reacts instantaneously with the reagent and dithiothreitol reacts slowly. The absorbance due to the coenzyme A-5,5'-dithiobis(2-nitrobenzoic acid) reaction is determined by extrapolating the linear (dithiol) absorbance-time curve to the time of addition of the reagent. After subtraction of control absorbance (deletion of NADPH), the concentration of CoA-SH is calculated from epsilon(max) = 1.36 x 10(4) at 412 nm. The method of protein removal and reduction of sulfhydryl groups on the enzyme are critical. This method provides an immediate assay. Recovery of reduced coenzyme A was 98.7%. The assay is applicable for microsomes or purified enzyme and has an effective range of 0.5-50 nmoles of coenzyme A. It was applied to kinetic measurement of the pigeon liver microsomal enzyme reaction. The apparent K(m) value for 3-hydroxy-3-methylglutaryl CoA was 1.75 x 10(-5) M, and for NADPH the value was 6.81 x 10(-4) M. This method was compared with the dual-label method at high and low levels of activity. The data were not statistically different.     

Rapid-scan stopped-flow studies of the pH dependence of the reaction between mercuric reductase and NADPH

The reaction of NADPH with the flavoenzyme mercuric reductase has been studied by rapid-scan stopped-flow spectrophotometry at 5 degrees C in the pH range 5.1-9.5. An intermediate formed within the dead time of the apparatus, and proposed to be an NADPH complex of oxidized enzyme, has an almost pH-independent spectrum. At pH 5.1 the formation of this species is followed by a rapid bleaching (k = 145 s-1) of the main flavin absorption band at 455 nm concomitantly with an absorbance increase around 395 nm. This process, which has a kinetic hydrogen isotope effect of 2.4, becomes less prominent at higher pH values and is not detectable above pH 7. It is suggested that this process includes the formation of a covalent thiol-flavin C-4a derivative stabilized by protonation of the active site. In the presence of an excess of NADPH, the final product of the reaction is probably an NADPH complex of two-electron-reduced enzyme, but below pH 6 the final spectrum becomes less intense suggesting a partial formation of four-electron-reduced enzyme. The spectral changes observed above pH 7 are nearly independent of pH. The first measurable step (k = 48 s-1 at pH 9.5) is thought to include the formation of an NADP+ complex of two-electron-reduced enzyme, while the final step (k = 6.3 s-1 at pH 9.5) results in the above-mentioned NADPH complex with two-electron-reduced enzyme. A minimal kinetic scheme rationalizing the observed pH dependence of the reaction and the observed isotope effects is presented.     

A new assay for L-aspartate:2-oxoglutarate aminotransferase.

A new enzymatic assay for aspartate aminotransferase is presented. The 2-oxoglutarate formed in transamination between l-glutamate and oxalacetate was determined in a system coupled with hydroxyglutarate dehydrogenase and NADH by following a decrease in absorbance at 340 nm. The method allowed accurate determination of the initial velocity of the reaction, which was proportional to the enzyme concentration. The Michaelis constants of pig heart cytosolic aspartate aminotransferase for l-glutamate and oxalacetate and the amino acceptor specificity using l-glutamate as an amino donor were determined. The method was applicable to the determination of the enzyme activity in various materials including rat serum and bacterial crude extract.     

Adrenodoxin reductase. Properties of the complexes of reduced enzyme with NADP+ and NADPH.

Anaerobic reduction of the flavoprotein adrenodoxin reductase with NADPH yields a spectrum with long wavelength absorbance, 750 nm and higher. No EPR signal is observed. This spectrum is produced by titration of oxidized adrenodoxin reductase with NADPH, or of dithionite-reduced adrenodoxin reductase with NADP+. Both titrations yield a sharp endpoint at 1 NADP(H) added per flavin. Reduction with other reductants, including dithionite, excess NADH, and catalytic NADP+ with an NADPH generating system, yields a typical fully reduced flavin spectrum, without long wavelength absorbance. The species formed on NADPH reduction appears to be a two-electron-containing complex, with a low dissociation constant, between reduced adrenodoxin reductase and NADP+, designated ARH2-NADP+. Titration of dithionite-reduced adrenodoxin reductase with NADPH also produces a distinctive spectrum, with a sharp endpoint at 1 NADPH added per reduced flavin, indicating formation of a four-electron-containing complex between reduced adrenodoxin reductase and NADPH. Titration of adrenodoxin reductase with NADH, instead of NADPH, provides a curved titration plot rather than the sharp break seen with NADPH, and permits calculation of a potential for the AR/ARH2 couple of -0.291 V, close to that of NAD(P)H (-0.316 V). Oxidized adrenodoxin reductase binds NADP+ much more weakly (Kdiss=1.4 X 10(-5) M) than does reduced adrenodoxin reductase, with a single binding site. The preferential binding of NADP+ to reduced enzyme permits prediction of a more positive oxidation-reduction potential of the flavoprotein in the presence of NADP+; a change of about + 0.1 V has been demonstrated by titration with safranine T. From this alteration in potential, a Kdiss of 1.0 X 10(-8) M for binding of NADP+ to reduced adrenodoxin reductase is calculated. It is concluded that the strong binding of NADP+ to reduced adrenodoxin reductase provides the thermodynamic driving force for formation of a fully reduced flavoprotein form under conditions wherein incomplete reduction would otherwise be expected. Stopped flow studies demonstrate that reduction of adrenodoxin reductase by equimolar NADPH to form the ARH2-NADP+ complex is first order (k=28 s-1). When a large excess of NADPH is used, a second apparently first order process is observed (k=4.25 s-1), which is interpreted as replacement of NADPH for NADP+ in the ARH2-NADP+ complex. Comparison of these rate constants to catalytic flavin turnover numbers for reduction of various oxidants by NADPH, suggests an ordered sequential mechanism in which reduction of oxidant is accomplished by the ARH2-NADP+ complex, followed by dissociation of NADP+. The absolute dependence of NADPH-cytochrome c reduction on both adrenodoxin reductase and adrenodoxin is confirmed...     

Preferential utilization of NADPH as the endogenous electron donor for NAD(P)H:quinone oxidoreductase 1 (NQO1) in intact pulmonary arterial endothelial cells

The goal was to determine whether endogenous cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) preferentially uses NADPH or NADH in intact pulmonary arterial endothelial cells in culture. The approach was to manipulate the redox status of the NADH/NAD(+) and NADPH/NADP(+) redox pairs in the cytosolic compartment using treatment conditions targeting glycolysis and the pentose phosphate pathway alone or with lactate, and to evaluate the impact on the intact cell NQO1 activity. Cells were treated with 2-deoxyglucose, iodoacetate, or epiandrosterone in the absence or presence of lactate, NQO1 activity was measured in intact cells using duroquinone as the electron acceptor, and pyridine nucleotide redox status was measured in total cell KOH extracts by high-performance liquid chromatography. 2-Deoxyglucose decreased NADH/NAD(+) and NADPH/NADP(+) ratios by 59 and 50%, respectively, and intact cell NQO1 activity by 74%; lactate restored NADH/NAD(+), but not NADPH/NADP(+) or NQO1 activity. Iodoacetate decreased NADH/NAD(+) but had no detectable effect on NADPH/NADP(+) or NQO1 activity. Epiandrosterone decreased NQO1 activity by 67%, and although epiandrosterone alone did not alter the NADPH/NADP(+) or NADH/NAD(+) ratio, when the NQO1 electron acceptor duroquinone was also present, NADPH/NADP(+) decreased by 84% with no impact on NADH/NAD(+). Duroquinone alone also decreased NADPH/NADP(+) but not NADH/NAD(+). The results suggest that NQO1 activity is more tightly coupled to the redox status of the NADPH/NADP(+) than NADH/NAD(+) redox pair, and that NADPH is the endogenous NQO1 electron donor. Parallel studies of pulmonary endothelial transplasma membrane electron transport (TPMET), another redox process that draws reducing equivalents from the cytosol, confirmed previous observations of a correlation with the NADH/NAD(+) ratio.     

Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase

Activity of the pterin- and folate-salvaging enzymes pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). Although this assay has been adequate to study the biology of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymatically coupled screening assay in which the product of the enzymatic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-molecule inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS.     

Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii

A method is described for the partial purification of pyridine nucleotide transhydrogenase from Azotobacter vinelandii (ATCC 9104) cells. The most highly purified preparation catalyzes the reduction of 300 mumoles of nicotinamide adenine dinucleotide (NAD(+)) per min per mg of protein under the assay conditions employed. The enzyme catalyzes the reduction of NAD(+), deamino-NAD(+), and thio-NAD(+) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as hydrogen donor, and the reduction of nicotinamide adenine dinucleotide phosphate (NADP(+)) and thio-NAD(+) with reduced NAD (NADH) as hydrogen donor. The reduction of acetylpyridine AD(+), pyridinealdehyde AD(+), acetylpyridine deamino AD(+), and pyridinealdehydedeamino AD(+) with NADPH as hydrogen donor was not catalyzed. The enzyme catalyzes the transfer of hydrogen more readily from NADPH than from NADH with different hydrogen acceptors. The transfer of hydrogen from NADH to NADP(+) and thio-NAD(+) was markedly stimulated by 2'-adenosine monophosphate (2'-AMP) and inhibited by adenosine diphosphate (ADP), adenosine triphosphate (ATP), and phosphate ions. The transfer of hydrogen from NADPH to NAD(+) was only slightly affected by phosphate ions and 2'-AMP, except at very high concentrations of the latter reagent. In addition, the transfer of hydrogen from NADPH to thio-NAD(+) was only slightly influenced by 2'-AMP, ADP, ATP, and other nucleotides. The kinetics of the transhydrogenase reactions which utilized thio-NAD(+) as hydrogen acceptor and NADH or NADPH as hydrogen donor were studied in some detail. The results suggest that there are distinct binding sites for NADH and NAD(+) and perhaps a third regulator site for NADP(+) or 2'-AMP. The heats of activation for the transhydrogenase reactions were determined. The properties of this enzyme are compared with those of other partially purified transhydrogenases with respect to the regulatory functions of 2'-AMP and other nucleotides on the direction of flow of hydrogen between NAD(+) and NADP(+).     

Purification and regulation of glucose-6-phosphate dehydrogenase from Bacillus licheniformis

d-Glucose-6-phosphate nicotinamide adenine dinucleotide phosphate (NADP) oxidoreductase (EC 1.1.1.49) from Bacillus licheniformis has been purified approximately 600-fold. The enzyme appears to be constitutive and exhibits activity with either oxidized NAD (NAD(+)) or oxidized NADP (NADP(+)) as electron acceptor. The enzyme has a pH optimum of 9.0 and has an absolute requirement for cations, either monovalent or divalent. The enzyme exhibits a K(m) of approximately 5 muM for NADP(+), 3 mM for NAD(+), and 0.2 mM for glucose-6-phosphate. Reduced NADP (NADPH) is a competitive inhibitor with respect to NADP(+) (K(m) = 10 muM). Phosphoenolpyruvate (K(m) = 1.6 mM), adenosine 5'-triphosphate (K(m) = 0.5 mM), adenosine diphosphate (K(m) = 1.5 mM), and adenosine 5'-monophosphate (K(m) = 3.0 mM) are competitive inhibitors with respect to NAD(+). The molecular weight as estimated from sucrose density centrifugation and molecular sieve chromatography is 1.1 x 10(5). Sodium dodecyl sulfate gel electrophoresis indicates that the enzyme is composed of two similar subunits of approximately 6 x 10(4) molecular weight. The intracellular levels of glucose-6-phosphate, NAD(+), and NADP(+) were measured and found to be approximately 1 mM, 0.9 mM, and 0.2 mM, respectively, during logarithmic growth. From a consideration of the substrate pool sizes and types of inhibitors, we conclude that this single constitutive enzyme may function in two roles in the cell-NADH production for energetics and NADPH production for reductive biosynthesis.     

Purification and characterization of ferredoxin-NAD(P)(+) reductase from the green sulfur bacterium Chlorobium tepidum

Ferredoxin-NAD(P)(+) reductase [EC 1.18.1.3, 1.18.1.2] was isolated from the green sulfur bacterium Chlorobium tepidum and purified to homogeneity. The molecular mass of the subunit is 42 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the native enzyme is approximately 90 kDa, estimated by gel-permeation chromatography, and is thus a homodimer. The enzyme contains one FAD per subunit and has absorption maxima at about 272, 385, and 466 nm. In the presence of ferredoxin (Fd) and reaction center (RC) complex from C. tepidum, it efficiently catalyzes photoreduction of both NADP(+) and NAD(+). When concentrations of NADP(+) exceeded 10 microM, NADP(+) photoreduction rates decreased with increased concentration. The inhibition by high concentrations of substrate was not observed with NAD(+). It also reduces 2,6-dichlorophenol-indophenol (DPIP) and molecular oxygen with either NADPH or NADH as efficient electron donors. It showed NADPH diaphorase activity about two times higher than NADH diaphorase activity in DPIP reduction assays at NAD(P)H concentrations less than 0.1 mM. At 0.5 mM NAD(P)H, the two activities were about the same, and at 1 mM, the former activity was slightly lower than the latter.     

Circular-dichroism studies of ligand binding to dihydrofolate reductase from Lactobacillus casei MTX/R.

Circular-dichroism spectra (200--450 nm) were recorded for Lactobacillus casei MTX/R dihydrofolate reductase and its complexes with substrates, inhibitors and coenzymes. These spectra are compared with those reported by others for dihydrofolate reductase from other sources. The binding of NADP+ or NADPH is associated with the perturbation of one or more aromatic amino acid residues, and there is marked enhancement of the negative c.d. band at 340 nm arising from the dihydronicotinamide chromophore of NADPH. The substrates folate and dihydrofolate give rise to substantial extrinsic c.d. bands on binding, which show a number of specific differences between enzymes from different sources. The binary complexes between the enzyme and the inhibitors methotrexate or trimethoprim also show strong c.d. bands, and these are qualitatively very similar for all dihydrofolate reductases studied so far. The ternary complexes between enzyme, NADPH and trimethoprim or methotrexate are very different from the sum of the spectra of the binary complexes. Trimethoprim leads to the disappearance of the 340 nm c.d. band of bound NADPH, whereas in the methotrexate--NADPH--enzyme ternary complex a "couplet" c.d. spectrum is observed at long wavelengths. Analysis of this latter feature suggests that it arises from a direct interaction between the dihydronicotinamide and pteridine rings in the ternary complex.     

Overall kinetic mechanism of saccharopine dehydrogenase (L-glutamate forming) from Saccharomyces cerevisiae

Kinetic studies were carried out for histidine-tagged saccharopine reductase from Saccharomyces cerevisiae at pH 7.0, suggesting a sequential mechanism with ordered addition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to the free enzyme followed by L-alpha-aminoadipate-delta-semialdehyde ( L-AASA) which adds in rapid equilibrium prior to l-glutamate in the forward reaction direction. In the reverse reaction direction, nicotinamide adenine dinucleotide phosphate (NADP) adds to the enzyme followed by addition of saccharopine. Product inhibition by NADP is competitive vs NADPH and noncompetitive vs alpha-AASA and L-glutamate, suggesting that the dinucleotide adds to the free enzyme prior to the aldehyde. Saccharopine is noncompetitive vs NADPH, alpha-AASA, and L-glutamate. In the direction of saccharopine oxidation, NADPH is competitive vs NADP and noncompetitive vs saccharopine, L-glutamate is noncompetitive vs both NADP and saccharopine, while L-AASA is noncompetitive vs saccharopine and uncompetitive vs NADP. The sequential mechanism is also corroborated by dead-end inhibition studies using analogues of AASA, L-glutamate, and saccharopine. 2-Amino-6-heptenoic acid was chosen as a dead-end analogue of L-AASA and is competitive vs AASA, uncompetitive vs NADPH, and noncompetitive vs L-glutamate. alpha-Ketoglutarate (alpha-Kg) serves as the dead-end analogue of L-glutamate and is competitive vs L-glutamate and uncompetitive vs L-AASA and NADPH. In the direction of saccharopine oxidation, N-oxalylglycine, L-pipecolic acid, L-leucine, alpha-ketoglutarate, glyoxylic acid, and L-ornithine were used as dead-end analogues of saccharopine and showed competitive inhibition vs saccharopine and uncompetitive inhibition vs NADP. The equilibrium constant for the reaction was measured at pH 7.0 by monitoring the change in absorbance of NADPH and is 200 M(-1). The value is in good agreement with the value determined using the Haldane relationship.     

Identification and characterization of the product release steps within the catalytic cycle of protochlorophyllide oxidoreductase

The chlorophyll biosynthetic enzyme protochlorophyllide reductase (POR) catalyzes the reduction of protochlorophyllide (Pchlide) into chlorophyllide (Chlide) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. POR is a light-driven enzyme, which has provided a unique opportunity to trap intermediates and identify different steps in the reaction pathway by initiating catalysis with illumination at low temperatures. In the present work we have used a thermophilic form of POR, which has an increased conformational rigidity at comparable temperatures, to dissect and study the final stages of the reaction where protein dynamics are proposed to play an important role in catalysis. Low-temperature fluorescence and absorbance measurements have been used to demonstrate that the reaction pathway for this enzyme consists of two additional "dark" steps, which have not been detected in previous studies. Product binding studies were used to show that spectroscopically distinct Chlide species could be observed and were dependent on whether the NADPH or NADP(+) cofactor was present. As a result we have been able to identify the intermediates that are observed during the latter stages of the POR catalytic cycle and have shown that they are formed via a series of ordered product release and cofactor binding events. These events involve release of NADP(+) from the enzyme and its replacement by NADPH, before release of the Chlide product has taken place. Following release of Chlide, the subsequent binding of Pchlide allows the next catalytic cycle to proceed.     

Characterization of a ferredoxin:NADP+ oxidoreductase from a nonphotosynthetic plant tissue

A flavoprotein with properties similar to those of ferredoxin:NADP+ oxidoreductases found in the leaves of higher plants has been purified to apparent homogeneity from bean sprouts, a nonphotosynthetic plant tissue. The absorbance and circular dichroism spectra of the bean sprout protein are similar to those of spinach leaf ferredoxin:NADP+ oxidoreductase and an antibody raised against the spinach enzyme recognized the bean sprout enzyme. The bean sprout enzyme catalyzed ferredoxin-dependent electron transfer from NADPH to equine cytochrome c at a high rate but, unlike the spinach enzyme, exhibited little NADPH to 2,6-dichlorophenol indophenol diaphorase activity. The bean sprout enzyme forms a 1:1 electrostatically stabilized complex with ferredoxins isolated from either bean sprouts or spinach leaves.