Stopped-flow fluorescence and steady-state kinetic studies of ligand-binding reactions of glucoamylase from Aspergillus niger.

The presteady-state and steady-state kinetics of the binding and hydrolysis of substrates, maltose and isomaltose, and the transition-state analogue, gluconolactone, by glucoamylase from Aspergillus niger were investigated using initial-rate, stopped-flow and steady-state methods. The change in the intrinsic fluorescence of the enzyme was monitored. Distinct mechanistic differences were observed in the interaction of the enzyme with maltose compared to isomaltose. Hydrolysis of maltose requires a three-step mechanism, whereas that of isomaltose involves at least one additional step. The rates of an observed conformational change, which is the second discernible step of the reactions, clearly show a tighter binding of maltose compared to isomaltose, probably because the reverse rate constants differ. Compared to the non-enzymic hydrolysis the transition-state stabilization energy of glucoamylase is approximately -66 kJ/mol with maltose and only -14 kJ/mol with isomaltose. Kinetic analysis of the binding of the inhibitor, gluconolactone, implies that independent interactions of two molecules occur. One of these, apparently, is a simple, fast association reaction in which gluconolactone is weakly bound. The other resembles binding of maltose, involving a fast association followed by a conformational change. Based on the results obtained, we propose new reaction mechanisms for Aspergillus glucoamylase.     

Peroxidase-catalyzed oxidation of capsaicinoids: steady-state and transient-state kinetic studies

Capsaicinoids are the pungent compounds in Capsicum fruits (i.e., "hot" peppers). Peroxidases catalyze capsaicinoid oxidation and may play a central role in their metabolism. However, key kinetic aspects of peroxidase-catalyzed capsaicinoid oxidation remain unresolved. Using transient-state methods, we evaluated horseradish peroxidase compound I and II reduction by two prominent capsaicinoids (25 degrees C, pH 7.0). We determined rate constants approaching 2 x 10(7) and 5 x 10(5)M(-1)s(-1) for compound I and compound II reduction, respectively. We also determined k(app) values for steady-state capsaicinoid oxidation approaching 8 x 10(5)M(-1)s(-1) (25 degrees C, pH 7.0). Accounting for stoichiometry, these are in excellent agreement with constants for compound II reduction, suggesting that this reaction governs capsaicinoid-dependent peroxidase turnover. Ascorbate rapidly reduced capsaicinoid radicals, assisting in the determination of the kinetic constants reported. Because ascorbate accumulates in Capsicum fruits, it may also be an important determinant for capsaicinoid content and preservation in Capsicum fruits and related products.     

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: pre-steady-state kinetic studies

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes the first step in the biosynthesis of the hypermodified A37 residue in tRNAs that read codons beginning with uridine. The mechanism of the enzyme-catalyzed reaction was studied by isotope trapping, pre-steady-state rapid quench, and single turnover experiments. Isotope trapping indicated that the enzyme.tRNA complex is catalytically competent, whereas the enzyme.DMAPP complex is not. The results are consistent with an ordered sequential mechanism for substrate binding where tRNA binds first. The association and dissociation rate constants for the enzyme.tRNA binary complex are 1. 15+/-0.33x10(7) M(-1) s(-1) and 0.06+/-0.01 s(-1), respectively. Addition of DMAPP gives an enzyme.tRNA.DMAPP ternary complex in rapid equilibrium with the binary complex and DMAPP. Rapid quench studies yielded a linear profile (k(cat)=0.36+/-0.01 s(-1)) with no evidence for buildup of enzyme-bound product. Product release from DMAPP-tRNA transferase is therefore not rate-limiting. The Michaelis constant for tRNA and the equilibrium dissociation constant for DMAPP calculated from the individual rate constants determined here are consistent with values obtained from a steady-state kinetic analysis.     

pH-induced conformational changes in spinach ferredoxin: steady-state and time-resolved fluorescence studies.

Steady-state and time-resolved fluorescence techniques were used to monitor pH-induced conformational changes in spinach ferredoxin. An increase was seen in the wave-length maximum of tryptophan-73 (Trp-73) emission, from 325 nm below pH 6.0 to 342 nm above pH 7.0, indicating significantly diminished hydrophobicity, at pH 7.0, in the environment of the indole ring. Raising the solution pH from 6.0 to 7.6 also decreased the binding of the detergent Brij-96, showing that the ferredoxin molecule as a whole became more hydrophilic at higher pH. Nonionic (acrylamide) and ionic (I- and Cs+) quenchers were used to probe the tryptophan environment. Trp-73 is partially shielded from I-, presumably by negatively charged residues, as predicted from the amino acid sequence and three-dimensional structure of plant-type ferredoxins. Ionic strength and pH effects on tryptophan fluorescence lifetimes follow a pattern common to single-tryptophan proteins: the emission decays can be fit to a biexponential model in which the lifetime of the excited state increases with increasing pH. The indication of a pH-induced conformational change in the range pH 6.0 to 7.6 is discussed with reference to the physiological association of ferredoxin with ferredoxin:NADP+ oxidoreductase and the rise in chloroplast stromal pH in the light.     

Vitamin K-dependent carboxylase from calf liver: studies on the steady-state kinetic mechanism

The steady-state kinetic mechanism of vitamin K-dependent carboxylase from calf liver has been investigated by initial-velocity measurements with varying concentrations of two carboxylase substrates and constant, nonsaturating concentrations of the other two substrates. With all combinations of the varied substrates tested linear kinetics were obtained with lines intersecting on the left side of the 1/v axis in double-reciprocal plots. Thus the carboxylase has a sequential reaction mechanism which includes the quinternary complex of the enzyme with its four substrates. A mechanism with the ordered steady-state addition of all substrates to the enzyme accords well with the results. A totally random mechanism was excluded but the alternative possibility remained that part of the substrates are added in a rapid-equilibrium random reaction. Experiments with saturating constant concentrations of sodium bicarbonate and varying concentrations of the other substrates suggest that bicarbonate (CO2) is either the first or, more probably, the last substrate bound to the enzyme.     

Resonance energy-transfer and fluorescence intensity studies of the transport of liposome-encapsulated molecules into isolated mouse liver nuclei

We present evidence that liposomes (composed of egg yolk L-alpha-phosphatidylcholine/phosphatidylethanolamine/cholesterol, in a molar ratio of 4:5:1) fuse with isolated mouse liver nuclei at low pH. Using the resonance energy-transfer assay, we determined the rate and extent of liposome and nuclear membrane lipid mixing. Fusion was substantial when the pH was below 5. The half-time of lipid mixing decreased by acidification of the solvent, reaching about 2 min at pH 4.5. In order to study the transport of the liposome-aqueous contents to the interior of the nuclei during the process, we developed fluorescence assays in which fluorescein isothiocyanate labeled dextrans of 150 kDa molecular mass (FITC-D150) were encapsulated in liposomes. These liposomes also included in their bilayers the fluorescent lipid N-tetramethylrhodamine-L-alpha-dipalmitoylphosphatidylethanolamine (N-Rh-DPPE). After incubation of these liposomes with mouse liver nuclei (pH 4.5, 37 degrees C, 30 min), we measured the fluorescence spectra of a suspension of washed nuclei and of nuclei treated by the detergent Triton X-100 (membrane-denuded nuclei). These Triton X-100 treated nuclei had no N-Rh-DPPE fluorescence while they showed a FITC-D150 fluorescence which amounted to 20% of that of the intact nuclei. In another assay, a laser beam was focused on single nuclei by a microscope epiexcitation device. The variation of the N-Rh-DPPE and FITC-D150 fluorescence with the nuclear radius was determined with the microphotometric attachment of the microscope.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methylenetetrahydrofolate reductase from Escherichia coli: elucidation of the kinetic mechanism by steady-state and rapid-reaction studies

The flavoprotein methylenetetrahydrofolate reductase (MTHFR) from Escherichia coli catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) using NADH as the source of reducing equivalents. The enzyme also catalyzes the transfer of reducing equivalents from NADH or CH(3)-H(4)folate to menadione, an artificial electron acceptor. Here, we have determined the midpoint potential of the enzyme-bound flavin to be -237 mV. We have examined the individual reductive and oxidative half-reactions constituting the enzyme's activities. In an anaerobic stopped-flow spectrophotometer, we have measured the rate constants of flavin reduction and oxidation occurring in each half-reaction and have compared these with the observed catalytic turnover numbers measured under steady-state conditions. We have shown that, in all cases, the half-reactions proceed at rates sufficiently fast to account for overall turnover, establishing that the enzyme is kinetically competent to catalyze these oxidoreductions by a ping-pong Bi-Bi mechanism. Reoxidation of the reduced flavin by CH(2)-H(4)folate is substantially rate limiting in the physiological NADH-CH(2)-H(4)folate oxidoreductase reaction. In the NADH-menadione oxidoreductase reaction, the reduction of the flavin by NADH is rate limiting as is the reduction of flavin by CH(3)-H(4)folate in the CH(3)-H(4)folate-menadione oxidoreductase reaction. We conclude that studies of individual half-reactions catalyzed by E. coli MTHFR may be used to probe mechanistic questions relevant to the overall oxidoreductase reactions.     

Methionyl-tRNA synthetase induced 3'-terminal and delocalized conformational transition in tRNAfMet: steady-state fluorescence of tRNA with a single fluorophore

Five species of tRNAfMet labeled with a single fluorophore are prepared to analyze the conformational changes at the 3'-end, at dihydrouridine, and at thiouridine in tRNAfMet upon binding of methionyl-tRNA synthetase. The emission and excitation spectra, anisotropy, and solvent accessibility of the fluorophore in each of the modified tRNAfMet's are determined in the absence and presence of methionyl-tRNA synthetase. The results are consistent with the following. The probes at the 3'-end are in a nonpolar environment, mobile relative to the tRNA molecule, and fully exposed to the solvent. The probes at dihydrouridine are partially stacked over the neighboring bases, nearly immobile, and relatively inaccessible. The S8-C13 cross-linked product is rigid. Upon binding of methionyl-tRNA synthetase, the probes at the 3'-terminus become localized in a less polar environment, highly immobilized, and effectively shielded against solvent access, while the probes at dihydrouridine appear to be partially unstacked from the neighboring base and become slightly more accessible for solvent. Singlet-singlet energy transfer between the intrinsic protein fluorescence and the fluorophores in modified tRNA's was observed by sensitized emission for tRNAfMet modified at the 3'-end and for S8-C13 but not for tRNAfMet's modified at dihydrouridine. These results suggest that dihydrouridine in tRNAfMet is oriented away from methionyl-tRNA synthetase in the tRNA-enzyme complex.     

The partially unfolded state of beta-momorcharin characterized with steady-state and time-resolved fluorescence studies

The specific conformation of partially unfolded state of beta-momorcharin was characterized through the steady-state and time-resolved fluorescence spectroscopic studies on a single Trp-190 which located adjacently to the active site. The content of secondary structure was retained, the binding of ANS was remarkably enhanced, and the correlation time of entire protein rotation was prolonged at the partially unfolded state formed by being equilibrated with the mild concentration of guanidine hydrochloride. The time-resolved fluorescence depolarization and excitation energy transfer analysis suggest that Trp-190 approached 2 A closer to Tyr-70 and was hidden from the exposure to the protein surface, while the rotational correlation time and freedom of its segmental motion were shortened and enhanced, respectively. These results suggest that the transient folding/unfolding intermediate state of beta-momorcharin adopt the specific conformation at the vicinity of the active site, although it exhibits very similar properties with those of the generally known molten-globule state.     

Picosecond fluorescence kinetic studies of electron acceptor Q redox heterogeneity

We have investigated the influence of chloroplast organization on the nature of chemical reductive titrations of Photosystem II fluorescence decay kinetics in spinach chloroplasts. Structural changes of the chloroplast membrane system were induced by varying the ionic environment of the thylakoids. A single-photon timing system with picosecond resolution monitored the kinetics of the chlorophyll a fluorescence emission. At all ionic concentrations studied, we have observed biphasic potentiometric titration curves of fluorescence yield; these have been interpreted to be suggestive of electron acceptor Q heterogeneity (Karukstis, K.K. and Sauer, K. (1983) Biochim. Biophys. Acta 722, 364–371; Cramer, W.A. and Butler, W.L. (1969) Biochim. Biophys. Acta 172, 503–510). A direct relation is observed between the E_m value of the low-potential component of Q and the Mg²⁺ concentration of the chloroplast suspending medium. We have attributed these midpoint potential variations to the thylakoid structural rearrangements involved in cation-regulated grana stacking. Ionic effects on the fluorescence decay kinetics at the redox transitions are discussed in terms of the heterogeneity of Photosystem II units (α- and β-centers) and the mechanism of deexcitation at a closed reaction center (fluorescence or nonradiative decay).     

Interaction of a fluorescent analogue of GDP with elongation factor Tu: steady-state and time-resolved fluorescence studies

A fluorescent derivative of GDP was prepared by the reaction of 2'-amino-2'-deoxy-GDP with fluorescamine. This derivative binds tightly (KD approximately 4.5 X 10(-8) M) to elongation factor Tu (EF-Tu) from Escherichia coli. The emission properties, including spectra, polarizations, and lifetimes, for fluorescamine-GDP free in solution and bound to EF-Tu are presented. Emission data on the fluorescamine-ethylamine conjugate are also given. A multifrequency phase and modulation lifetime study (using nine modulation frequencies over the range of 2-80 MHz) indicated that the emissions of these three systems were well characterized by single exponential decays corresponding to 1.45 ns for the fluorescamine-ethylamine derivative in buffer and to 7.74 and 11.03 ns for the fluorescamine-GDP derivative free in buffer and bound to EF-Tu, respectively. Multifrequency differential polarized phase fluorometry results indicated a rotational relaxation time of the protein-probe complex of approximately 88 ns; these data also indicated the lack of significant local motion for the probe. Addition of excess GDP to the EF-Tu-probe complex led to displacement of the fluorescamine-GDP derivative as evidenced by the change in both the steady-state and dynamic polarization values. The observed increase in fluorescence intensity upon displacement allowed us to follow the kinetics of the dissociation reaction; a dissociation rate constant of 5.0 X 10(-3) S-1 was determined. These results demonstrate the utility of this 2'-amino-2'-deoxy-GDP analogue as a probe of guanosine nucleotide dependent systems.     

Electron spin resonance and steady-state fluorescence polarization studies of lipid bilayers containing integral proteins

We derive equations that describe changes in the steady-state fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene (DPH) or in the spectrum of electron spin resonance (ESR) nitroxide spin-labeled lipid probes as a function of the intrinsic molecule concentration in lipid bilayer membranes. We make use of an assumption used by us in an earlier paper. The equations are independent of any membrane model. They are valid when a DPH probe or a spin-labeled chain is equivalent to an unlabeled lipid hydrocarbon chain only as far as their general space-filling properties are concerned. We consider cases where the bilayer is either in a single homogeneous phase or in a two-phase region. We apply our equations to analyze ESR data from delipidated sarcoplasmic reticulum membranes and from egg yolk phosphatidylcholine bilayers containing Ca2+-ATPase, and DPH data from dipalmitoylphosphatidylcholine (DPPC) bilayers containing Ca2+-ATPase, both for T greater than Tc. The following conclusions were derived: (i) Ca2+-ATPase oligomers are "randomly" distributed, for the concentrations studied, in the fluid phase. (ii) There is no fixed stoichiometric ratio of "boundary" lipids and oligomers. (iii) Between 24k and 28k lipid molecules are able to surround each isolated oligomer composed of k Ca2+-ATPase monomers. Finally, we apply our equations to analyze DPH studies on DPPC bilayers containing Ca2+-ATPase for T less than Tc. We find that the results reported are in accord with the predictions of the model. In the Appendix, we show that an analytical expression for probabilities used by us is in very good agreement with the results of computer simulation.     

Catalytic mechanism of biotin carboxylase: steady-state kinetic investigations

Biotin carboxylase was purified from Escherichia coli by a new procedure, and its steady-state kinetic parameters were examined. MgATP and bicarbonate add to the enzyme randomly, followed by addition of biotin. Both bicarbonate and MgATP add in rapid equilibrium. A catalytic base with a pK of 6.6 is observed in V/K profiles. Inactivation studies also revealed a sulfhydryl group in the active site that is essential for catalysis. It is proposed that the acid-base catalysts are necessary for the tautomerization of biotin, which presumably enhances its nucleophilicity toward the carboxyl group donor. A second enzymic group with a pK of 6.6, whose role is unknown, is seen in Vmax profiles. The pH profiles for the biotin carboxylase catalyzed phosphorylation of ADP by carbamoyl phosphate have the same shape as the profiles for the forward reaction, which demonstrates that the enzymic bases assume the same protonation states for catalysis of transphosphorylation in either direction. The lack of reactivity of thionucleotide analogues of ATP when Mg is used as the divalent metal ion suggests that both metal ions required for reaction coordinate to the nucleotide. The second metal ion appears to be absolutely required for reaction and not merely an activator of the reaction. Characterization of a bicabonate-dependent biotin-independent ATPase activity strongly suggests that carboxylation proceeds via a carboxyphosphate intermediate.     

tRNA selection and kinetic proofreading in translation

Using single-molecule methods we observed the stepwise movement of aminoacyl-tRNA (aa-tRNA) into the ribosome during selection and kinetic proofreading using single-molecule fluorescence resonance energy transfer (smFRET). Intermediate states in the pathway of tRNA delivery were observed using antibiotics and nonhydrolyzable GTP analogs. We identified three unambiguous FRET states corresponding to initial codon recognition, GTPase-activated and fully accommodated states. The antibiotic tetracycline blocks progression of aa-tRNA from the initial codon recognition state, whereas cleavage of the sarcin-ricin loop impedes progression from the GTPase-activated state. Our data support a model in which ribosomal recognition of correct codon-anticodon pairs drives rotational movement of the incoming complex of EF-Tu-GTP-aa-tRNA toward peptidyl-tRNA during selection on the ribosome. We propose a mechanistic model of initial selection and proofreading.     

Design, synthesis, and spectroscopic properties of peptide-bridged fluorescence energy-transfer cassettes

A general partial solid-phase synthetic scheme was developed for the synthesis of energy-transfer cassettes with the donor and acceptor dyes bridged by a peptide. In these cassettes, 6-carboxyfluorescein (Fam) served as a donor. For the second dye, 6-carboxy-X-rhodamine (Rox) was used as a fluorescent acceptor or erythrosin B as a quencher. Different peptides bearing Rox at the amino terminus and Fam linked through different diamines to the carboxyl terminus were synthesized to examine the effects of the chain length and rigidity on energy-transfer efficiency. The ratio of emission intensities at 605 nm of the acceptor dye (ROX) in the cassette Rox-GPPPEPPP-p-xylylenediamine-Fam versus free ROX with 488 nm excitation was approximately 14 and is similar to that obtained for optimized oligonucleotide primers bearing the same dyes [Ju, J., Ruan, C., Fuller, C. W., Glazer, A. N., and Mathies, R. A. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 4347-4351].     

L-Ornithine carbamoyltransferase from Saccharomyces cerevisiae: steady-state kinetic analysis.

Ornithine carbamoyltransferase of Saccharomyces cerevisiae is subjected to an enzymatic regulation of its anabolic activity when it is bound to the inducible catabolic arginase as described earlier. This regulatory ornithine carbamoyltransferase essentially catalyzes the synthesis of citrulline, but the reverse reaction could be demonstrated using arsenate instead of phosphate. Steady-state initial velocity studies of the reverse reaction indicate that the mechanism is consistent with a rapid-equilibrium random model (in which all steps are in equilibrium, except that concerned with the interconversion of the central ternary complexes) involving the formation of enzyme - ornithine - arsenate and enzyme - citrulline - phosphate dead-end complexes. In the forward direction, although the mechanism also appears to be random, the results are in better agreement with a preferred ordered binding of substrates, with carbamoylphosphate adding first. This degenerate form of the random mechanism is discussed.     

Multiple ion-dependent and substrate-dependent Na+/K+-ATPase conformational states. Transient and steady-state kinetic studies

The hydrolysis of beta-(2-furyl)acryloyl phosphate (FAP), catalyzed by the Na+/K+-ATPase, is faster than the catalyzed hydrolysis of ATP. This is due to catalyzed hydrolysis of the pseudosubstrate by K+-dependent states of the enzyme, thus bypassing the Na+-dependent enzyme states that are required and are rate limiting in ATP hydrolysis. Unlike ATP, FAP is a positive effector of the E2 state. A study of FAP hydrolysis permits a detailed analysis of later steps in the overall ion translocation-ATP hydrolysis pathway. During the steady state of FAP hydrolysis in the presence of K+, substantial phosphoryl-enzyme is formed, as is indicated by the covalent incorporation of 32P from [32P]FAP. A comparison of the phosphoryl-enzyme yield with the rate of overall hydrolysis reveals that at 25 degrees C the phosphoryl-enzyme formed is all kinetically competent. Both the yield of phosphoryl-enzyme and the rate of overall hydrolysis of FAP are [K+] dependent. The transition E1 in equilibrium E2 is also [K+] dependent, but the rate of transition is differently affected by [K+] than are the above-mentioned two processes. Two distinct roles for K+ are indicated, as an effector of the E1-E2 equilibrium and as a "catalyst" in the hydrolysis of the E2-P. In contrast to the results at 25 degrees C, a virtually stoichiometric yield of phosphoryl-enzyme occurs at 0 degree C in the presence of Na+ and the absence of K+. At lower concentrations of K+ and in the presence of Na+, the hydrolysis of FAP at 0 degree C proceeds substantially through the E1-E2 pathway characteristic of ATP hydrolysis. The selectivity of FAP for the E2-K+-dependent pathway is due to the thermal inactivation of E1 at 25 degrees C in the absence of ATP or ATP analogues, even at high concentrations of Na+. These results emphasize the existence of multiple functional "E1" and "E2" states in the overall ATPase-ion translocation pathway.     

Binding of camphor to Pseudomonas putida cytochrome p450(cam): steady-state and picosecond time-resolved fluorescence studies

The binding of camphor to cytochrome P450(cam) has been investigated by steady-state and time-resolved tryptophan fluorescence spectroscopy to obtain information on the substrate access channel. The fluorescence quenching experiments show that some of the tryptophan residues undergo changes in their local environment on camphor binding. The time-resolved fluorescence decay profile gives four lifetime components in the range from 99 ps to 4.5 ns. The shortest lifetime component assigned to W42 lies close to the proposed camphor access channel. The results show that the fluorescence of W42 is greatly affected on binding of camphor, and supports dynamic fluctuations involved in the passage of camphor through the access channel as proposed earlier on the basis of crystallographic, molecular dynamics simulation and site-directed mutagenesis studies.