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S C Tu

Publications and source records attributed to S C Tu.

At least 55 records · Page 3Linked to original sources
PubMed1989

Chemical modification and characterization of the alpha cysteine 106 at the Vibrio harveyi luciferase active center.

Vibrio harveyi luciferase, an alpha beta dimer, was effectively inactivated by treatment with the methylation agent methyl p-nitrobenzene sulfonate. However, inactivation of luciferase in the presence of excess amounts of this reagent did not follow pseudo-first-order kinetics. After taking the autodecay of this reagent into consideration in kinetic analysis, the pseudo-first-order constants and subsequently the second-order rate constant (83 min-1 M-1 at pH 7 and 23 degrees C) were determined. The inactivation rate can be retarded by the addition of the decanal or the reduced FMN substrate but not by the reaction product FMN. The binding of decanal specifically protected one target residue against modification with a concomitant protection of luciferase against inactivation. A pentapeptide containing this specific target residue was isolated and identified to be Phe-Gly-Ile-X-Arg with X corresponding to the S-methylated form of the cysteinyl residue at position 106 of the luciferase alpha subunit. It is concluded that this reactive alpha Cys-106 is at the aldehyde site and is also near the reduced flavin site of luciferase. The modified enzyme exhibited no gross conformational changes detectable by protein fluorescence measurements, which may be due to the small size change of the target cysteinyl residue after methylation. The methylated enzyme still retained the ability to bind one decanal and one reduced FMN without any substantial changes in binding affinities. The cause of luciferase inactivation by the methylation of alpha Cys-106 has been shown to be the impaired ability to form the 4a-hydroperoxy-flavin intermediate from the bound flavin substrate or to stabilize this intermediate.

Amino Acid Sequence↗
PubMed1988

Delineation of bacterial luciferase aldehyde site by bifunctional labeling reagents.

Previously we have established that a highly reactive cysteinyl group on the alpha subunit is at the aldehyde site of the (alpha beta) dimeric Vibrio harveyi luciferase. Three isomeric bifunctional reagents have been synthesized and used to further delineate the luciferase aldehyde site. These probes differ in their relative positions of and distances between the two functional groups active in chemical and photochemical labelings, respectively. Each of the probes can effectively and reversibly inactivate luciferase by forming a disulfide linkage primarily to the reactive cysteinyl residue. Upon subsequent photolysis, a diazoacetate arm in each probe was activated for photochemical labeling of amino acid residues within reach. After reductive regeneration of the reactive cysteinyl residue, 0.35-0.40 probe per dimeric luciferase was found to have been photochemically incorporated, correlating well with the degree of irreversible enzyme inactivation. Low but significant amounts of the three isomeric probes initially attached to the alpha reactive cysteine through a disulfide have been found to photochemically tag certain residues on beta. The latter residues are estimated to be no more than 8-11 A away from the alpha reactive cysteine. Thus the reactive cysteinyl residue, and hence the aldehyde site, must be at or near the alpha beta subunit interface. Furthermore, the structural integrity of the microenvironment surrounding this reactive cysteinyl residue is crucial to luciferase activity. An HPLC method for the isolation of luciferase alpha and beta subunits has also been developed.

Affinity Labels↗
PubMed1988

Studies of electron-transfer properties of salicylate hydroxylase from Pseudomonas cepacia and effects of salicylate and benzoate binding.

The pH dependence of the redox behavior of salicylate hydroxylase from Pseudomonas cepacia as well as the effects of salicylate, benzoate, and chloride binding is described. At pH 7.6 in 0.02 M potassium phosphate buffer E1(0')(EFl ox/EFl.-) is -0.150 V and E2(0')(EFl.-/EFl red H-) is -0.040 V versus the standard hydrogen electrode (SHE). A maximum of 5% of FAD anion semiquinone is thermodynamically stabilized under these conditions. However, in coulometric and dithionite titrations more semiquinone is kinetically formed, indicating slow transfer of the second electron. The potential/pH dependence is consistent with a two-electron, one-proton transfer. Upon salicylate binding the midpoint potential is shifted 0.020 V negative from -0.094 to -0.114 V vs SHE at pH 7.6. A maximum of 7% of the neutral semiquinone is stabilized both in potentiometric and coulometric titrations. This small potential shift indicates that the substrate is bound nearly to the same extent to all three oxidation states of the enzyme. It is clear that the substrate binding does not make the reduction of the flavin thermodynamically more favorable. In contrast to salicylate, the potential shift caused by the effector, benzoate, is much more significant. (A maximum potential shift of -0.07 V is calculated.) Benzoate binds most tightly to the oxidized form and is least tightly bound to the two-electron-reduced form of the enzyme. For the reduction of the free enzyme the transfer of the second electron or the transfer of the proton is rate limiting, as is shown by the kinetic formation of the anionic semiquinone.(ABSTRACT TRUNCATED AT 250 WORDS)

Benzoates↗
PubMed1987

Pseudomonas cepacia 3-hydroxybenzoate 6-hydroxylase: induction, purification, and characterization.

A single strain of Pseudomonas cepacia cells was differentially induced to synthesize salicylate hydroxylase, 3-hydroxybenzoate 6-hydroxylase, or 4-hydroxybenzoate 3-hydroxylase. A procedure was developed for the purification of 3-hydroxybenzoate 6-hydroxylase to apparent homogeneity. The purified hydroxylase appears to be a monomer with a molecular weight of about 44,000 and exhibits optimal activity near pH 8. The hydroxylase contains one FAD per enzyme molecule and utilizes NADH and NADPH with similar efficiencies. The reaction stoichiometry for this enzyme has been determined. In comparison with other aromatic flavohydroxylases, this enzyme is unique in inserting a new hydroxyl group to the substrate at a position para to an existing one.

Enzyme Induction↗
PubMed1987

Pseudomonas cepacia 3-hydroxybenzoate 6-hydroxylase: stereochemistry, isotope effects, and kinetic mechanism.

A neutral flavin semiquinone species was formed upon photoreduction of Pseudomonas cepacia 3-hydroxybenzoate 6-hydroxylase whereas no flavin radical was detected by anaerobic reduction with NADH in the presence of m-hydroxybenzoate. In the latter case, the formation of flavin semiquinone is apparently thermodynamically unfavorable. A stereospecificity for the abstraction of the 4R-position hydrogen of NADH has been demonstrated for this hydroxylase. Deuterium and tritium isotope effects were observed with (4R)-[4-2H]NADH and (4R)-[4-3H]NADH as substrates. The DV effect indicates the existence of at least one slow step after the isotope-sensitive enzyme reduction by dihydropyridine nucleotide. A minimal kinetic mechanism has been deduced on the basis of initial velocity measurements and studies on deuterium and tritium isotope effects. Following this scheme, m-hydroxybenzoate and NADH bind to the hydroxylase in a random sequence. The flavohydroxylase is reduced by NADH, and NAD+ is released. Oxygen subsequently binds to and reacts with the reduced flavohydroxylase-m-hydroxybenzoate complex. Following the formation and release of water and gentisate, the oxidized holoenzyme is regenerated. The enzyme has a small (approximately 2-fold) preference for the release of NADH over m-hydroxybenzoate from the enzyme-substrates ternary complex.

Anaerobiosis↗
PubMed1986

Kinetic analysis of enzyme inactivation by an autodecaying reagent.

A simple method is described for the determination of both the pseudo-first-order rate constant and the second-order rate constant for enzyme inactivation by a chemical reagent which itself undergoes exponential decay. The validity of this method has been demonstrated in two test cases in which the labile diethyl pyrocarbonate was used to inactivate salicylate hydroxylase and bacterial luciferase.

Diethyl Pyrocarbonate↗
PubMed1985

Isolation and characterization of a cyclic AMP receptor protein from luminous Vibrio harveyi cells.

A cAMP receptor protein (CRP) species was purified from the luminous Vibrio harveyi cells to apparent homogeneity. This protein had a dimeric structure with a molecular weight of 23,000 per subunit. Among all eight nucleotides tested, only cAMP (Kd = 3 to 4 microM at 0 degrees C and 52 microM at 23 degrees C) and cGMP (Kd = 6 to 10 microM at 0 degrees C and 67 microM at 23 degrees C) bound to this protein. Its binding to poly(dI-dC), poly(dA-dT), and DNA fragments isolated from V. harveyi cells were all significantly enhanced by the addition of cAMP. Based on patterns of limited proteolysis by trypsin, this CRP assumes different conformations in the absence and presence of cAMP. Also consistent with this conclusion is the finding that the binding of cAMP to CRP induced about 50% quenching of the CRP fluorescence with a concomitant 3-nm blue shift from the original 336-nm emission peak. The binding of cGMP resulted in similar fluorescence changes but had no apparent effect on the pattern of proteolysis by trypsin. Using an in vitro transcription system known to be dependent on cAMP and Escherichia coli CRP, the synthesis of a run-off transcript product was also significantly enhanced by cAMP and this V. harveyi CRP.

Cyclic AMP↗
PubMed1984

The kinetic mechanism of salicylate hydroxylase as studied by initial rate measurement, rapid reaction kinetics, and isotope effects.

The kinetic mechanism of Pseudomonas cepacia salicylate hydroxylase has been examined by steady state initial rate measurements, and stopped flow and equilibrium studies. Results indicate that salicylate and NADH bind to the hydroxylase randomly. The enzyme is reduced and NAD+ is released. Oxygen subsequently binds to the reduced enzyme . substrate complex, leading to the production of hydroxylated product, CO2, and water. Based on results of anaerobic rapid mixing experiments, the rate of enzyme reduction by NADH is enhanced 290- and 240-fold when the hydroxylase is complexed with salicylate and benzoate (a nonsubstrate effector), respectively. Salicylate enhances, whereas benzoate slightly weakens, the NADH binding to the enzyme. Primary isotope effects were observed with (4R)-[4-2H]- and (4R)-[4-3H]NADH but not with the (4S)-[4-2H]NADH. Using varying concentrations of benzoate, the pattern of tritium isotope effect on Vm/Km, T(V/K), also indicates that benzoate and NADH bind to the enzyme randomly. The intrinsic isotope effects, Dk, of (4R)-[4-2H]NADH on the reduction of enzyme . salicylate and enzyme . benzoate complexes were found to be 5.57 and 5.96, respectively. The former is much repressed but the latter is only slightly so in the expression of their corresponding deuterium isotope effects on Vm, DV. Furthermore, values of DV (1.69 to 5.07) show a rough correlation with the extents of uncoupling of substrate hydroxylation and H2O2 formation activities for a series of benzenoid effectors. These results indicate that relative to the step of enzyme reduction, the subsequent reaction(s) leading to H2O2 formation must be fast whereas that for substrate hydroxylation contains at least one slow step.

Anaerobiosis↗
PubMed1984

Affinity labeling of the aldehyde site of bacterial luciferase.

2-Bromo[1-14C]1-decanal was synthesized as an affinity labeling probe for the aliphatic aldehyde site of Vibrio harveyi luciferase. In the presence of excess amounts of this probe, the inactivation of bacterial luciferase occurred following apparent first order kinetics. This inactivation was markedly retarded in the presence of decanal but neither butanal (a very poor aldehyde substrate) nor FMN (a reaction product derived from reduced FMN) showed any significant protective effect. Upon mixing luciferase with the affinity labeling probe, a noncovalent complex was formed prior to the covalent attachment. At pH 6 and 23 degrees C, the dissociation constant for the binding step and the rate constant for the covalent modification step were determined to be 23 microM and 1 min-1, respectively. The displacement of a bound aldehyde substrate by this probe added secondarily was also demonstrated. The inactivation of luciferase was correlated with both the incorporation of about 1.2 molecules of the probe and the loss of 0.8 to 1.1 cysteinyl residues/luciferase alpha beta dimer. The presence of an essential sulfhydryl group at the aldehyde site of luciferase has thus been demonstrated. This sulfhydryl group was a constituent residue of the alpha subunit and was near the alpha beta subunit interface. This residue appears to be the same essential cysteinyl group previously identified by chemical modification (Nicoli, M.Z., Meighen, E.A., and Hastings, J.W. (1974) J. Biol. Chem. 249, 2385-2392). The labeled luciferase did not exhibit any significant binding for the reduced FMN substrate.

Affinity Labels↗
PubMed1984

Apoenzyme of Pseudomonas cepacia salicylate hydroxylase. Preparation, fluorescence property, and nature of flavin binding.

The apoenzyme of Pseudomonas cepacia salicylate hydroxylase was prepared by a dialysis method. The apoprotein retains a dimeric structure and binds one FAD per monomer. Flavin binding results in both 81 and 60% of quenching and 15- and 5-nm blue shifts of FAD and protein fluorescence, respectively. A hydrophobic environment for the flavin site and a conformational difference between apoprotein and holoenzyme are thus indicated. Prior binding of NADH markedly retards the holoenzyme activity development upon a subsequent FAD addition. Flavin 1,N6-ethenoadenine dinucleotide binds to the apoenzyme much more weakly than FAD but this reconstituted holoenzyme and the FAD X enzyme both exhibit similar activities. The adenine moiety appears to be important to binding. The formation of holoenzyme from apoprotein and FAD involves minimally a two-step reversible process, an initial flavin-binding step followed by a conformational transition. At both 6 and 23 degrees C, the rates of hydroxylase activity recovery can be correlated with the rates of FAD binding, indicating that the initial FAD X apoenzyme complex is fully active and the subsequent slow conformational change has no significant effect on the catalytic efficiency. Overall dissociation constants calculated based on kinetic data are essentially identical with those determined by equilibrium measurements.

Apoenzymes↗
PubMed1983

Characterization of the aldehyde binding site of bacterial luciferase by photoaffinity labeling.

A photoaffinity probe 1-diazo-2-oxoundecane has been synthesized and used to examine the aldehyde-binding site of the nonidentical dimeric luciferase (alpha beta) from Vibrio harveyi cells. In the dark, the probe competes against aldehyde in binding to luciferase. Irradiation of luciferase and the probe at 254 nm resulted in primarily specific labeling of both alpha and beta subunits with concomitant enzyme inactivation, but significant (congruent to 40%) nonspecific labeling of mainly the beta subunit also occurred. The addition of decanal to protect the active center reduced the rate of inactivation. When 2-mercaptoethanol was included to quench the nonspecific labeling, the amounts of probe incorporated into alpha and beta correlated stoichiometrically with the quantities of enzyme photoinactivated. On the basis of these findings, we postulate that the aldehyde binding site is at or near the subunit interface of luciferase.

Affinity Labels↗
PubMed1981

Determination of the position of monooxygenation in the formation of catechol catalyzed by salicylate hydroxylase.

[3,5-2H]Salicylate was obtained by a hydrogen/deuterium exchange reaction carried out in 2H2O. Using this deuterated salicylate as a substrate for salicylate hydroxylase, the product catechol was isolated and characterized with respect to the positions of deuterium retention. Based on nuclear magnetic resonance and mass spectroscopy analyses, the product was identified as [3,5-2H]catechol. It is thus unequivocally demonstrated that salicylate is decarboxylated and hydroxylated at the same ring position. Also consistent with this conclusion is the lack of a kinetic isotope effect for the deuterated salicylate substrate.

Catechols↗