Biomedical subjects

P F Cook

Publications and source records attributed to P F Cook.

At least 37 records · Page 2Linked to original sources

PubMed1998

Glutamate 190 is a general acid catalyst in the 6-phosphogluconate-dehydrogenase-catalyzed reaction.

Site-directed mutagenesis was used to change E190 of sheep liver 6-phosphogluconate dehydrogenase to A, D, H, K, Q, and R to probe its possible role as a general acid catalyst. Each of the mutant proteins was characterized with respect to the pH dependence of kinetic parameters. Mutations that eliminate a titrable group at position 190, result in pH-rate profiles with no observable pK on the basic side of the V/K6PG profile. Mutations that change the pK of the group at position 190 result in the expected pK perturbations in the V/K6PG profile. Kinetic parameters obtained at the pH optimum in the pH-rate profiles are consistent with a rate-limiting tautomerization of the 1,2-enediol of ribulose 5-phosphate consistent with the proposed role of E190. Data are also consistent with some participation of E190 in an isomerization required to form the active Michaelis complex.

Amino Acid Sequence↗

PubMed1998

Multiple isotope effects as a probe of proton and hydride transfer in the 6-phosphogluconate dehydrogenase reaction.

Primary solvent deuterium, primary substrate deuterium, multiple solvent deuterium/substrate deuterium, and multiple solvent deuterium/13C isotope effects on V/K6PG have been measured for the Candida utilis and sheep liver 6-phosphogluconate dehydrogenases (6PGDH). Proton inventory data suggest the presence of a significant medium effect in a step preceding hydride transfer and the presence of a kinetic solvent deuterium isotope effect on hydride transfer. Multiple isotope effect data confirm the presence of multiple solvent deuterium sensitive steps, likely including a conformational change preceding hydride transfer, hydride transfer, and decarboxylation.

Animals↗

PubMed1998

Oxidative decarboxylation of 6-phosphogluconate by 6-phosphogluconate dehydrogenase proceeds by a stepwise mechanism with NADP and APADP as oxidants.

Primary kinetic deuterium, 13C, and multiple deuterium/13C-isotope effects on V/K6PG have been measured for the Candida utilis (cu) and sheep liver (sl) 6-phosphogluconate dehydrogenases (6PGDH). With NADP as the dinucleotide substrate, the following values of D(V/K6PG), 13(V/K6PG)H, and 13(V/K6PG)D were measured at pH 8 for cu6PGDH (sl6PGDH): 1.57 +/- 0.08 (1.87 +/- 0.10), 1.0209 +/- 0.0005 (1.0059 +/- 0.000 10), 1.0158 +/- 0.0001 (1.0036 +/- 0.0008). With APADP as the dinucleotide substrate, values for the above isotope effects at pH 8 are as follows: 2.98 +/- 0.08 (2.47 +/- 0.06), 1. 0106 +/- 0.0002 (1.0086 +/- 0.000 09), and 0.9934 +/- 0.0003 (0.9950 +/- 0.0003). Results indicate the oxidative decarboxylation of 6PG to the 1,2-enediol of ribulose 5-phosphate proceeds via a stepwise mechanism with hydride transfer preceding decarboxylation in all cases. The inverse 13C-isotope effect observed with APADP and 6PG-3d may reflect a preequlibrium isotope effect on the binding of 6PG preceding hydride transfer. Deuterium-isotope effects on V, V/KNADP, and V/K6PG are identical at all pHs and for both enzymes. The primary deuterium-isotope effect on V/K6PG for both enzymes is constant at pH values below the pK in the pH profile for V/K6PG, and decreases as the pH increases. Data suggest the development of rate limitation by a step or steps other than the hydride-transfer step as the pH is increased.

Animals↗

PubMed1998

Cysteine 42 is important for maintaining an integral active site for O-acetylserine sulfhydrylase resulting in the stabilization of the alpha-aminoacrylate intermediate.

O-Acetylserine sulfhydrylase-A (OASS-A) is a pyridoxal 5'-phosphate (PLP) dependent enzyme from Salmonella typhimurium that catalyzes the beta-replacement of acetate in O-acetyl-L-serine (OAS) by sulfide to give L-cysteine. The reaction occurs via a ping-pong kinetic mechanism in which alpha-aminoacrylate in Schiff base with the active site PLP is an intermediate [Cook, P. F., Hara, S., Nalabolu, S. R., and Schnackerz, K. D. (1992) Biochemistry 31, 2298-2303]. The sequence around the Schiff base lysine (K41) has been determined [Rege, V. D., Kredich, N. M., Tai, C.-H., Karsten, W. E., Schnackerz, K. D., & Cook, P. F. (1996) Biochemistry 35, 13485-13493], and the sole cysteine in the primary structure is immediately C-terminal to the lysine. In an effort to assess the role of C42, it has been changed to serine and alanine by site-directed mutagenesis. The mutant proteins are structurally nearly identical to the wild-type enzyme on the basis of UV-visible, fluorescence, far-UV and cofactor-induced CD, and 31P NMR studies, but subtle structural differences are noted. Kinetic properties of both mutant proteins differ significantly from those of the wild-type enzyme. The C42S mutant exhibits a > 50-fold increase in the OAS:acetate lyase activity and a 17-fold decrease in V for the cysteine synthesis compared to the wild-type enzyme, while decreases of > 200-fold in the OAS: acetate lyase activity and a 30-fold decrease in V for the cysteine synthesis are found for the C42A mutant enzyme. In both cases, however, the pH dependence of kinetic parameters for cysteine synthesis and OAS: acetate lyase activity yield, within error, identical pK values. In the three-dimensional structure of OASS-A, cysteine 42 is located behind the cofactor, pointing away from the active site, toward the interior of the protein. The dramatic change in the OAS:acetate lyase activity of OASS-A in the C42S and C42A mutant proteins likely results from a localized movement of the serine hydroxyl (compared to the cysteine thiol) toward additional hydrophilic, hydrogen-bonding groups in C42S, or away from hydrophilic groups for C42A, repositioning structure around and including K41. Subtle movement of the epsilon-amino group of K41 may change the geometry for nucleophilic displacement of the amino acid from PLP, leading to changes in overall activity and stability of the alpha-aminoacrylate intermediate. Data indicate that single amino acid substitutions that yield only subtle changes in structure can produce large differences in reaction rates and overall mechanism.

Alanine↗

PubMed1998

Secondary tritium and solvent deuterium isotope effects as a probe of the reaction catalyzed by porcine recombinant dihydropyrimidine dehydrogenase.

Dihydropyrimidine dehydrogenase catalyzes the rate-limiting step in the degradation of pyrimidines in mammals, the reduction of uracil or thymine to their 5,6-dihydro derivatives. The reduction of uracil by enzyme-bound reduced flavin involves both proton and hydride transfer. In order to determine whether hydride and proton transfer occur in a concerted or stepwise fashion, and to determine the nature of the transition state for the reduction, secondary tritium kinetic isotope effects were measured in H2O and D2O. The tritium isotope effect using 5-3H-uracil is 0.90 +/- 0.03 in H2O and becomes more inverse, 0.85 +/- 0.04, in D2O. Data are interpreted in terms of a stepwise reduction at C-6 followed by protonation at C-5. A late transition state is proposed for the proton transfer at C-5 of uracil.

Animals↗

PubMed1998

Three-dimensional structure of O-acetylserine sulfhydrylase from Salmonella typhimurium.

The last step in cysteine biosynthesis in enteric bacteria is catalyzed by the pyridoxal 5'-phosphate-dependent enzyme O-acetylserine sulfhydrylase. Here we report the crystal structure at 2.2 A resolution of the A-isozyme of O-acetylserine sulfhydrylase isolated from Salmonella typhimurium. O-acetylserine sulfhydrylase shares the same fold with tryptophan synthase-beta from Salmonella typhimurium but the sequence identity level is below 20%. There are some major structural differences: the loops providing the interface to the alpha-subunit in tryptophan synthase-beta and two surface helices of tryptophan synthase-beta are missing in O-acetylserine sulfhydrylase. The hydrophobic channel for indole transport from the alpha to the beta active site of tryptophan synthase-beta is, not unexpectedly, also absent in O-acetylserine sulfhydrylase. The dimer interface, on the other hand, is more or less conserved in the two enzymes. The active site cleft of O-acetylserine sulfhydrylase is wider and therefore more exposed to the solvent. A possible binding site for the substrate O-acetylserine is discussed.

Binding Sites↗

PubMed1998

Catalytic competence of O-acetylserine sulfhydrylase in the crystal probed by polarized absorption microspectrophotometry.

The reactions of the pyridoxal 5'-phosphate-dependent enzyme O-acetylserine sulfhydrylase with the substrate O-acetyl-L-serine and substrate analogs have been investigated in the crystalline state by single-crystal polarized absorption microspectrophotometry. This approach has allowed us to examine the catalytic competence of the enzyme in different crystalline states, one of which was used to determine the three-dimensional structure; experimental conditions were defined for the accumulation of catalytic intermediates in the crystal suitable for crystallographic analyses.O-Acetyl-L-serine reacts with the enzyme in one of the crystal forms leading via a beta-elimination reaction to the accumulation of the alpha-aminoacrylate Schiff base, absorbing maximally at 320 and 470 nm, as in solution. The dissociation constant for the alpha-aminoacrylate Schiff base is in the millimolar range, 500-fold higher than in solution, suggesting that crystal lattice interactions may oppose functionally relevant conformational changes. The dissociation constant exhibits a bell-shaped dependence on pH centered at pH 7. At this pH the alpha-aminoacrylate species slowly decays with time (30% decrease in 24 hours). The alpha-aminoacrylate intermediate readily reacts with sodium azide, an analog of sulfide, the natural nucleophilic agent, to give a new amino acid and the native enzyme, indicating that the crystalline enzyme catalyzes the overall beta-replacement reaction as in solution. In other crystal forms, including that used for the X-ray investigation, O-acetyl-L-serine either has an even higher dissociation constant or causes crystal damage upon binding. When the crystalline enzyme reacts with either L-cysteine or L-serine, the external aldimine intermediate is formed. The dissociation constants for both substrate analogs are closer to those observed in solution and are modulated by pH as in solution. Findings demonstrate that O-acetylserine sulfhydrylase is catalytically competent in the crystal although some regions of the molecule, likely involved in an open-closed transition induced by O-acetyl-L-serine binding, may have a limited flexibility. The accumulation in the crystal of both the external aldimine and the alpha-aminoacrylate intermediate makes feasible their structural determination and, therefore, the elucidation of the catalytic pathway at the molecular level.

Alanine↗

PubMed1998

Cloning, expression, purification, and characterization of the 6-phosphogluconate dehydrogenase from sheep liver.

The mRNA encoding the 51-kDa subunit of 6-phosphogluconate dehydrogenase (6PGDH) from sheep liver was reverse-transcribed and amplified. The resulting cDNA was reamplified in N-terminal and C-terminal segments and spliced to generate a full-length clone, and an internal cDNA fragment was also amplified. The full-length clone containing the complete coding sequence of the 6PGDH cDNA was sequenced and found to contain two mutations and two deletions in the internal region and two mutations outside of the internal region, an A to G point mutation at position 1407 that resulted in the amino acid change Gln 445 to Arg and a silent mutation at position 1426. The internal clone was sequenced and shown to be free of any mutations; therefore the internal piece was used to replace the same region in the full-length clone to correct the mutations in this region. The mutation at position 1407 which was outside of the internal region was corrected using site-directed mutagenesis. The cDNA with the correct codon was then subcloned into the bacterial expression vector pQE-30 and overproduced in Escherichia coli strain M15. A protein with a subunit molecular weight of 51,000 was expressed at a level of about 4.5% of the total soluble protein in M15 as judged by SDS/PAGE. Cloning into pQE-30 adds six histidines and a short linker to the N-terminus of the enzyme. The recombinant 6PGDH with His-tag was purified using the Ni-NTA affinity column supplied by Qiagen. The purification procedure resulted in a homogeneous protein by SDS/PAGE with 22.4-fold purification with an overall yield of 61%. The recombinant enzyme exhibits kinetic parameters within error identical to those measured for native sheep liver enzyme.

Animals↗

PubMed1998

Mechanism from isotope effects.

Isotope effects represent perhaps one of the most versatile tools available to investigators interested in the determination of reaction mechanism, particularly in the case of the mechanistic enzymologist. Interpretation of isotope effect data is somewhat more difficult for enzyme reactions, since the chemical or isotope-dependent step(s) is(are) normally not solely rate-limiting as they are for non-enzyme-catalyzed reactions. One can, however, take advantage of rate-limitation by multiple steps in an enzyme-catalyzed reaction to obtain information on a number of aspects of mechanism. In this paper, simple theory for the application of isotope effects to reaction mechanism is developed, and applied to organic reactions and those catalyzed by enzymes. Techniques used to measure isotope effects depend somewhat on the isotope used, that is radioisotope vs. stable isotope, or hydrogen isotope vs. heavier atoms. Techniques to be discussed include competitive and noncompetitive (or internal discrimination) measurements. In enzyme-catalyzed reactions, information can be obtained on the order of addition of reactants and relase of products, and this will be illustrated using the 6-phosphogluconate and alcohol dehydrogenase reactions. The use of multiple isotope effects can be used to distinguish between stepwise and concerted reactions, and this will be illustrated with the formate and glucose 6-phosphate dehydrogenase and malic enzyme reactions.

Alcohol Dehydrogenase↗

PubMed1997

Time-resolved fluorescence of O-acetylserine sulfhydrylase catalytic intermediates.

The reaction of the substrate O-acetyl-L-serine (OAS) with the pyridoxal 5'-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase-A (OASS-A) proceeds via the transient formation of an external aldimine absorbing at 420 nm and a stable alpha-aminoacrylate intermediate absorbing at 330 and 465 nm. Stable external aldimine species are obtained by reaction of the enzyme with either the reaction product L-cysteine or the product analog L-serine. Static and time-resolved fluorescence emission properties of the coenzyme in the above catalytic intermediates have been used to directly probe the active site conformation at different stages of the catalytic pathway. Upon excitation at either 420 or 330 nm, the external aldimines with L-cysteine and L-serine exhibit a structured emission centered at 490 nm with a shoulder at 530 nm. Fluorescence decays upon excitation at 420 nm are best fitted using two components with lifetimes of 1.1 and 3.8 ns, with the fractional intensity of the slow component being 0.92 with L-cysteine and 0.75 with L-serine, respectively. The fast component, emitting at 530 nm, is attributed to a dipolar species formed in the excited state by proton dissociation, and the slow component, emitting at 490 nm, is attributed to a ketoenamine tautomer of the external aldimine. The slow component for external aldimine fluorescence decay is characterized by the same lifetime value as that of the internal aldimine with an increased fractional intensity, indicating that the distribution between the ketoenamine and the dipolar species is shifted toward the ketoenamine tautomer in the external aldimine, compared to the internal aldimine. Differences in equilibrium distribution of ketoenamine and enolimine tautomers can also account for differences in the emission properties of the external aldimines of L-cysteine and L-serine. The alpha-aminoacrylate species is characterized by a relatively weak emission. Upon excitation at 330 nm, the emission exhibits two bands centered at 420 and 540 nm, whereas upon excitation at 420 nm the emission bands are centered at 500 and 540 nm, and upon excitation at 465 nm, the main absorbance peak of the alpha-aminoacrylate species, the emission spectrum shows a band at 540 nm. The fluorescence decays, upon excitation at 330 nm, are best fitted using three components with lifetime values similar to those found for the internal aldimine, with the slow component predominating. Species-associated spectra, collected between 400 and 520 nm upon excitation at 350 nm, indicate the presence of a fast component overlapping the slow component on the blue side of the emission spectrum, as detected for the internal aldimine. When the excitation wavelength is 420 nm, there are only two components with the fast one predominating. A further increase in the fractional intensity of the fast component is observed upon excitation at 465 nm. The weak emission and the short lifetime of the emission excited at 465 nm indicate that this alpha-aminoacrylate tautomer interacts significantly with neighboring groups of the protein matrix and may be endowed with a higher mobility than the external aldimine.

Catalysis↗

PubMed1997

Chemical mechanism of the fructose-6-phosphate,2-kinase reaction from the pH dependence of kinetic parameters of site-directed mutants of active site basic residues.

A bifunctional enzyme, fructose-6-phosphate 2-kinase-fructose 2, 6-bisphosphatase, catalyzes synthesis and degradation of fructose 2, 6-bisphosphate. Mutants of basic residues, including Lys51, Arg78, Arg79, Arg136, Lys172, and Arg193, immediately around the active site of rat testis fructose 6-P,2-kinase were constructed, and their steady state kinetics, ATP binding, and the effect of pH on the kinetics were characterized. All mutants showed a several-fold increase in KMgATP, much larger increases in KFru 6-P, and decreased V compared to those of the wild type enzyme (WT). Replacement of Lys172 and Arg193 with Ala and Leu, respectively, also produced mutants with large KFru 6-P values. Substitution of Lys51, which is located in a Walker-A motif (GXXGXGKT, amino acids 45-52), with Ala or His resulted in enzymes with increased KMgATP values and unable to bind Fru 6-P. The dissociation constants for 2'(3')-O-(N-methylanthraniloyl)-ATP (mantATP) and ATP of all these mutants except Lys51 were similar. Lys51 mutants were unable to bind mantATP. The pH dependence of V and the V/Ks for MgATP and Fru 6-P suggest a mechanism in which reactants and enzyme combine irrespective of the protonation state of groups required for binding and catalysis, but only the correctly protonated enzyme-substrate complex is catalytically active. A chemical mechanism is suggested in which a general base accepts a proton from the 2-hydroxyl of Fru 6-P concomitant with nucleophilic attack on the gamma-phosphate of MgATP. Phosphoryl transfer is also facilitated by interaction of the gamma-phosphate with a positively charged residue that neutralizes the remaining negative charge. The dianionic form of the 6-phosphate of fructose 6-P is required for binding, and it is likely anchored by a positively charged enzyme residue. A comparison of the pH dependence of kinetic parameters for Ala or His mutant proteins at Lys51, Lys172, and Arg79 suggests that Lys51 interacts with the gamma-phosphate of MgATP and that several other arginines likely participate in transition state stabilization of the transferred phosphoryl. The active site general base has yet to be identified.

Adenosine Triphosphate↗

PubMed1997

Expression, purification, and characterization of the recombinant NAD-malic enzyme from Ascaris suum.

The cDNA encoding the 65-kDa subunit of malic enzyme from Ascaris suum was cloned into the bacterial expression vector pKK223-3 and overproduced in Escherichia coli. A protein with a subunit molecular mass of 65,000 was expressed at a level of up to 3% of the total soluble protein in JM109, as judged by SDS-PAGE. The enzyme was purified using column chromatography on phenyl-Sepharose followed by orange-A agarose. The purification procedure resulted in a 32-fold purification with an overall yield of 51%. The bacterially expressed enzyme exhibits kinetic constants identical to those measured for native A. suum NAD-malic enzyme.

Animals↗

PubMed1997

Purification, characterization, and kinetics of porcine recombinant dihydropyrimidine dehydrogenase.

Porcine recombinant dihydropyrimidine dehydrogenase was purified from Escherichia coli cells using cell disruption, ammonium sulfate fractionation, and chromatography on DEAE-cellulose and 2',5'-ADP-Sepharose. The yield was 60% with a specific activity of 14 units/mg protein. On SDS/PAGE the purified dehydrogenase exhibits a single band, indicating that no proteolytic degradation was taking place during purification. In agreement with the native enzyme, all cofactors, FMN, FAD, NADPH, and two iron-sulfur clusters, have been found. EPR spectra of the reduced dehydrogenase obtained at pH 9.5 are characteristic for two [4Fe-4S]1+ cubanes in dipolar interaction. Quantification of the observed signals indicated 0.95 spins per subunit, showing only partially reduced iron-sulfur clusters. The kinetic parameters of the porcine recombinant enzyme are very similar to those of the native enzyme. Thus, it can be concluded that the porcine recombinant enzyme behaves like the native dehydrogenase.

Animals↗

PubMed1996

Kinetic and chemical mechanisms of the sheep liver 6-phosphogluconate dehydrogenase.

A complete kinetic characterization of sheep liver 6-phosphogluconate dehydrogenase including product and dead-end inhibition patterns, primary deuterium isotope effects, and the pH dependence of kinetic parameters has been completed in order to determine the kinetic mechanism and obtain information on the chemical mechanism of the enzyme. A rapid equilibrium random kinetic mechanism has been proposed, with product and dead-end inhibition patterns both being symmetric. Ribulose 5-phosphate and 6-sulfogluconate are both competitive with 6-phosphogluconate (6-PG) and noncompetitive with NADP, and NADPH and ATP-ribose are both competitive with NADP and noncompetitive with 6-phosphogluconate. Equal primary deuterium isotope effects of 1.5-2 on DV, DV/KNADP, and DV/K6-PG with 3-deuterio-6-PG confirm a rapid equilibrium random mechanism and show that hydride transfer is at least partially rate limiting in the overall reaction. The maximum velocity is pH dependent, decreasing at low and high pH with slopes of 1 and -1, respectively, and pK values of 6.4 and 8.6. The V/KNADP and V/K6-PG also decrease at low and high pH with slopes of 1 and -1, giving pK values of 6.8 and 8.7 and of 6.9 and 7.8, respectively. The pH rate profiles are consistent with a general acid/general base mechanism where the catalytic residues are involved in binding. Reverse protonation states between the general acid and the general base are proposed where an unprotonated general base accepts a proton from the C-3 hydroxyl of 6-PG concomitant with hydride transfer followed by decarboxylation of the resulting 3-keto intermediate to give an enediol which is protonated by the general acid to form ribulose 5-phosphate. The pH dependence of the pKi profile of the inhibitory analog 5-phosphoribonate decreases at low and high pH with slopes of 1 and -1, respectively, and pKs of 6.2 and 7.4 and suggests that intrinsic pKs are observed in the V/K profiles. The pKs of both the general base and general acid in the E:6-PG complex appears to be perturbed such that the general base decreases from 7.4-7.8 to a value of 6.4-6.8, and the pK of the general acid increases from 6. 2-6.9 to a value of 8.6-8.7, as a result of direct interaction with 6PG. Data are interpreted with regard to the published crystal structures of the E:6-PG, E:NADP, and E:NADPH complexes.

Animals↗

PubMed1996

Substitution of pyridoxal 5'-phosphate in the O-acetylserine sulfhydrylase from Salmonella typhimurium by cofactor analogs provides a test of the mechanism proposed for formation of the alpha-aminoacrylate intermediate.

O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the final step in the de novo synthesis of L-cysteine in Salmonella typhimurium. Complementary cofactor mutagenesis in which the active site PLP is substituted with cofactor analogs is used to test the mechanism proposed for the OASS. Data obtained with the pyridoxal 5'-deoxymethylenephosphonate-substituted enzyme suggest that the binding of OAS as it forms the external Schiff base is such that the acetate side chain is properly positioned for elimination (orthogonal to the developing alpha,beta-double bond) only about 1% of the time. Data support the assignment of an enzyme group with a pK of 6.7 that interacts with the acetyl side chain, maintaining it orthogonal to the developing alpha,beta-double bond. Similar studies of the 2'-methylpyridoxal 5'-phosphate-substituted enzyme suggest that, although the mechanism is identical to that catalyzed by native OASS, the reaction coordinate for alpha-proton abstraction may be decreased compared with that observed for the native enzyme.

Alanine↗

PubMed1996

A change in the internal aldimine lysine (K42) in O-acetylserine sulfhydrylase to alanine indicates its importance in transimination and as a general base catalyst.

O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5'-phosphate dependent enzyme that catalyzes a beta-replacement reaction forming L-cysteine and acetate from O-acetyl-L-serine (OAS) and sulfide. The pyridoxal 5'-phosphate (PLP) is bound at the active site in Schiff base linkage with a lysine. In the present study, the Schiff base lysine was identified as lysine 42, and its role in the OASS reaction was determined by changing it to alanine using site-directed mutagenesis. K42A-OASS is isolated as an external aldimine with methionine or leucine and shows no reaction with the natural substrates. Apo-K42A-OASS can be reconstituted with PLP, suggesting that K42 is not necessary for cofactor binding and formation of the external Schiff base. The apo-K42A-OASS, reconstituted with PLP, shows slow formation of the external aldimine but does not form the alpha-aminoacrylate intermediate on addition of OAS, suggesting that K42 is involved in the abstraction of the alpha-proton in the beta-elimination reaction. The external aldimines formed upon addition of L-Ala or L-Ser are stable and represent a tautomer that absorbs maximally at 420 nm, while L-Cys gives a tautomeric form of the external aldimine that absorbs at 330 nm, and is also seen in the overall reaction after addition of primary amines to the assay system. The use of a small primary amine such as ethylamine or bromoethylamine in the assay system leads to the initial formation of an internal (gamma-thialysine) or external (ethylamine) aldimine followed by the slow formation of the alpha-aminoacrylate intermediate on addition of OAS. Activity could not be fully recovered, and only a single turnover is observed. Data suggest a significant rate enhancement resulting from the presence of K42 for transimination and general base catalysis.

Alanine↗

PubMed1996

Purification and characterization of dihydropyrimidine dehydrogenase from Alcaligenes eutrophus.

Dihydropyrimidine dehydrogenase from Alcaligenes eutrophus was purified to homogeneity using ammonium sulfate fractionation and chromatography on phenyl-Sepharose, MonoQ-Sepharose, and 2,5-ADP-Sepharose. The enzyme is a homotetramer with a subunit molecular mass of 52 kDa. The absorption spectrum of the bacterial dihydropyrimidine dehydrogenase has maxima in the 300- and 400-nm region, suggesting a flavoprotein. The enzyme contains 4 mol FMN, about 24 mol iron and acidlabile sulfide per mole of protein, implying a flavoprotein with FeS centers. The bacterial dehydrogenase is NADPH dependent with B-side stereospecificity. The initial velocity patterns of the bacterial dehydrogenase together with isotope exchange at equilibrium and a quantitative analysis of the product and dead-end inhibition data suggest a rapid equilibrium random kinetic mechanism, which is in contrast to results obtained for dihydropyrimidine dehydrogenase from pig liver. The pig liver enzyme adheres to a nonclassical two-site ping-pong kinetic mechanism [B. Podschun, P. F. Cook, and K. D. Schnackerz (1990) J. Biol. Chem. 265, 12966-12972], whereas for the bovine enzyme a rapid equilibrium random kinetic mechanism was proposed based on steady-state kinetic data [D. J. T. Porter and T. Spector (1993) J. Biol. Chem. 268, 19321-19327].

Alcaligenes↗

PubMed1996

Tryptophan luminescence as a probe of enzyme conformation along the O-acetylserine sulfhydrylase reaction pathway.

O-Acetylserine sulfhydrylase A (OASS-A) is a pyridoxal 5'-phosphate- (PLP-) dependent enzyme that catalyzes the last step in the synthesis of L-cysteine, the beta-replacement of acetate in O-acetyl-L-serine (OAS) by sulfide. The phosphorescence properties of the two tryptophans of wild-type OASS-A, W51 and W162, and of W162 in the W51Y mutant protein have been characterized over the temperature range 170-273 K. In glasses at 170 K, the apoenzyme exhibits a phosphorescence spectrum which is the superposition of two spectra with well-resolved 0,0 vibronic bands centered at 405 and 410 nm, the blue lambda max suggesting that one of the two Trp residues in OASS-A is in a polar pocket, while the other is in a relatively hydrophobic pocket. The presence of PLP in the OASS-A holoenzyme reduces the intrinsic fluorescence by 40-45%, but the spectrum is unaltered except for the appearance of the internal Schiff base ketoenamine fluorescence band centered at 484 nm. The phosphorescence is strongly quenched by PLP, with about 70% reduction in intensity and lifetime. Further, the phosphorescence spectrum of the holoprotein exhibits a single and narrow 0,0 vibronic band centered at 405 nm and a broad band in the 450-550-nm range resulting from delayed fluorescence of the ketoenamine tautomer of the internal Schiff base, sensitized by triplet-singlet energy transfer from tryptophan to the ketoenamine tautomer of PLP. Comparison with data obtained for the W51Y mutant strongly suggests that the 405-nm phosphorescence band derives from W162, and that W51 in the wild type is entirely quenched either by singlet or triplet energy transfer to PLP or by some local group in the protein. From the rate of energy transfer, the separation between W162 and PLP is estimated to be about 25 A. Substrates other than OAS affect only the intensity of the coenzyme fluorescence band (484 nm) and the intensity of delayed fluorescence relative to that of phosphorescence, effects that are attributable to changes in fluorescence quantum yield of the ketoenamine chromophore. Addition of OAS, on the other hand, leads to a splitting of the 0,0 vibronic band in the phosphorescence spectrum of W162, yielding poorly resolved peaks at 406 and 408.5 nm, indicating thereby a change in the environment of the tryptophan residue and therefore in the conformation of the macromolecule as the internal Schiff base is converted to the alpha-aminoacrylate Schiff base. In buffer at 273 K, both the fluorescence and phosphorescence spectra relax to longer wavelengths and the phosphorescence lifetime is reduced to a few milliseconds, all indications that W162 is in a flexible region of the macromolecule, probably in close proximity to the aqueous interface. The phosphorescence lifetime in fluid medium reveals conformational heterogeneity in OASS-A and unveils important structure modulating effects of cofactor, substrates, and pH. Binding of PLP to the apoprotein increases the rigidity of the polypeptide in the region of W162 (in agreement with the greater thermal stability of the holoprotein), while OAS and L-serine have an opposite effect. Increasing the pH from 6.5 to 9 results in a 1.7-fold increase in tau av and a change in the relative amplitudes of the two lifetime components. Since the phosphorescence originates from a single tryptophan residue, the two tau components reflect distinct conformations of the subunit. In this case the conformational equilibrium (slow on the phosphorescence time scale) is governed by one or more groups in the protein with a pK around 8.

Cold Temperature↗