Rapid development of polyclonal antisera against infectious salmon anaemia virus and its optimization and application as a diagnostic tool.

Infectious salmon anaemia is an important disease of Atlantic salmon. One of the current methods of diagnosis is the indirect fluorescent antibody test (IFAT), using a monoclonal antibody specific to the haemagglutinin of the virus. The conformationally dependent nature of this antibody could be a drawback in its usefulness in other tests. This study describes the development and optimization of a polyclonal antiserum against infectious salmon anaemia virus, including a method of separating virus from cell culture components within culture supernatant. The antiserum was subsequently optimized for use in a variety of immunological diagnostic tests, including IFAT and an alkaline phosphatase-based immunoassay, and Western blot.

Characterization of the allosteric anion-binding site of O-acetylserine sulfhydrylase.

A new crystal structure of the A-isozyme of O-acetylserine sulfhydrylase-A (OASS) with chloride bound to an allosteric site located at the dimer interface has recently been determined [Burkhard, P., Tai, C.-H., Jansonius, J. N., and Cook, P. F. (2000) J. Mol. Biol. 303, 279-286]. Data have been obtained from steady state and presteady-state kinetic studies and from UV-visible spectral studies to characterize the allosteric anion-binding site. Data obtained with chloride and sulfate as inhibitors indicate the following: (i) chloride and sulfate prevent the formation of the external aldimines with L-cysteine or L-serine; (ii) chloride and sulfate increase the external aldimine dissociation constants for O-acetyl-L-serine, L-methionine, and 5-oxo-L-norleucine; (iii) chloride and sulfate bind to the allosteric site in the internal aldimine and alpha-aminoacrylate external aldimine forms of OASS; (iv) sulfate also binds to the active site. Sulfide behaves in a manner identical to chloride and sulfate in preventing the formation of the L-serine external aldimine. The binding of chloride to the allosteric site is pH independent over the pH range 7-9, suggesting no ionizable enzyme side chains ionize over this pH range. Inhibition by sulfide is potent (K(d) is 25 microM at pH 8) suggesting that SH(-) is the physiologic inhibitory species.

Pyridoxal 5'-phosphate-dependent alpha,beta-elimination reactions: mechanism of O-acetylserine sulfhydrylase.

O-Acetylserine sulfhydrylase catalyzes the replacement of the beta-acetoxy group of O-acetyl-L-serine with sulfide to generate L-cysteine. The reaction represents the final step in the biosynthesis of L-cysteine in enteric bacteria and plants. A quinonoid intermediate has not been detected using a variety of kinetic and spectroscopic probes for the wild-type or mutant enzymes, ruling out an E1 mechanism. The structure of the external Schiff base intermediate indicates an anti elimination. O-Acetylserine sulfhydrylase is the only known pyridoxal 5'-phosphate-dependent enzyme that catalyzes a beta-elimination reaction to have an anti E2 mechanism.

Identification of an allosteric anion-binding site on O-acetylserine sulfhydrylase: structure of the enzyme with chloride bound.

A new crystal structure of O-acetylserine sulfhydrylase (OASS) has been solved with chloride bound at an allosteric site and sulfate bound at the active site. The bound anions result in a new "inhibited" conformation, that differs from the "open" native or "closed" external aldimine conformations. The allosteric site is located at the OASS dimer interface. The new inhibited structure involves a change in the position of the "moveable domain" (residues 87-131) to a location that differs from that in the open or closed forms. Formation of the external aldimine with substrate is stabilized by interaction of the alpha-carboxyl group of the substrate with a substrate-binding loop that is part of the moveable domain. The inhibited conformation prevents the substrate-binding loop from interacting with the alpha-carboxyl group, and hinders formation of the external Schiff base and thus subsequent chemistry. Chloride may be an analog of sulfide, the physiological inhibitor. Finally, these results suggest that OASS represents a new class of PLP-dependent enzymes that is regulated by small anions.

O-acetylserine sulfhydrylase.

The 31P NMR data suggest slight differences in the structures around the 5'-P for the internal Schiff base and the lanthionine external Schiff base (both largely ketoeneamine) and a large difference for enolimine portion of the serine external Schiff base. Addition of cysteine or serine increase delayed fluorescence and triplet to singlet energy transfer. Addition of OAS exhibits a splitting of the 0,0 vibronic, the result of two distinct conformations, likely enolimine and ketoeneamine tautomers. Nonetheless, the alpha-amino-acrylate Schiff base conformation differs from either the internal or external Schiff base conformations. All of the time-resolved fluorescence data are consistent with conformation changes reflecting redistribution of ketoeneamine and enolimine tautomers as catalysis occurs. It is important to remember that the structural changes are substantial. The native structure (internal Schiff base) is active site open, while the K41A mutant enzyme (ketoeneamine external Schiff base) is active site closed. The trigger for the conformational change from open to closed as one goes from the internal to external Schiff base is the occupancy of the alpha-carboxyl subsite of the active site (Burkhard et al., 1999). Associated with this, as observed in pH-rate profiles, pH-dependent changes in phosphorescence, and pH-dependent changes in fluorescence enhancement upon binding acetate or cysteine is an enzyme group with a pK in the range 7-8. Dependent on the protonation state of the enzyme group, structural changes likely occur that also reflect a redistribution of the tautomeric equilibrium. Finally, the minimal catalytic cycle can likely be pictured as shown in Fig. 20. The changes may be pH dependent, and the open conformations for the internal Schiff base and the alpha-aminoacrylate Schiff base are not identical structurally, as expected because of the increased stability of the latter.

Ligand binding induces a large conformational change in O-acetylserine sulfhydrylase from Salmonella typhimurium.

Covalent binding of L-methionine as an external aldimine to the pyridoxal 5'-phosphate-cofactor in the K41A mutant of O-acetylserine sulfhydrylase from Salmonella typhimurium induces a large conformational change in the protein. Methionine mimics the action of the substrate O-acetyl-L-serine during catalysis. The alpha-carboxylate moiety of L-methionine in external aldimine linkage with the active site pyridoxal 5'-phosphate forms a hydrogen bonding network to the "asparagine-loop" P67-T68-N69-G70 which adopts a different conformation than in the native protein. The side-chain nitrogen of Asn69 moves more than 7 A to make a hydrogen bond to the alpha-carboxylate group of the inhibitor. As the external aldimine is formed, the PLP tilts by 13 degrees along its longitudinal axis such that C4' moves toward the entrance to the active site and the side-chain of the methionine is directed toward the active site entrance. The local rearrangement acts as a trigger to induce a large global conformational change in the protein. A subdomain comprised of beta-strand 4, alpha-helix 3, beta-strand 5 and alpha-helix 4 moves towards the active site by a rotation of 7 degrees. This subdomain movement results in a reduction of the severe twist of its central beta-sheet and reduces the active site entrance to a small hole, giving access only to small molecules like sulfide, the second substrate, or acetate, the first product.

Kinetic characterization of a T-state of Ascaris suum phosphofructokinase with heterotropic negative cooperativity by ATP eliminated.

The affinity analogue, 2',3'-dialdehyde ATP has been used to chemically modify the ATP-inhibitory site of Ascaris suum phosphofructokinase, thereby locking the enzyme into a less active T-state. This enzyme form has a maximum velocity that is 10% that of the native enzyme in the direction of fructose 6-phosphate (F6P) phosphorylation. The enzyme displays sigmoid saturation for the substrate fructose 6-phosphate (S0.5 (F6P) = 19 mM and nH = 2.2) at pH 6.8 and a hyperbolic saturation curve for MgATP with a Km identical to that for the native enzyme. The allosteric effectors, fructose 2,6-bisphosphate and AMP, do not affect the S0.5 for F6P but produce a slight (1.5- and 2-fold, respectively) V-type activation with Ka values (effector concentration required for half-maximal activation) of 0.40 and 0.24 mM, respectively. Their activating effects are additive and not synergistic. The kinetic mechanism for the modified enzyme is steady-state-ordered with MgATP as the first substrate and MgADP as the last product to be released from the enzyme surface. The decrease in V and V/K values for the reactants likely results from a decrease in the equilibrium constant for the isomerization of the E:MgATP binary complex, thus favoring an unisomerized form. The V and V/KF6P are pH dependent with similar pK values of about 7 on the acid side and 9.8 on the basic side. The microenvironment of the active site appears to be affected minimally as evidenced by the similarity of the pK values for the groups involved in the binding site for F6P in the modified and native enzymes.

Time-resolved fluorescence of O-acetylserine sulfhydrylase.

Static and time-resolved fluorescence of the internal aldimine of the pyridoxal 5'-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase (OASS) and those of free PLP, and the PLP-L-valine Schiff base have been measured to gain insight into the photophysics of PLP bound to OASS. Exciting at 330 nm, free coenzyme exhibits a band at 415 nm, whereas PLP-valine and OASS (also when excited at their absorbance maxima) exhibit a structured emission with a peak at 420 nm and shoulders at 490 and 530 nm. The emission bands at 420 and 490 nm are attributed to the enolimine and ketoenamine tautomers of the internal aldimine, respectively, while the 530 nm emission might arise from a dipolar species formed upon proton dissociation in the excited state. Time-resolved fluorescence of OASS (PLP-valine), excited at 412 nm (415 nm) and collected at lamda > 470 nm, indicates the presence of two components characterized by lifetimes (tau) of 0.6 (0.08) and 3.8 (1.55) ns with equal fractional intensity (f). In the presence of acetate the slow component dominates OASS emission with f of 0.98. Excitation at 350 nm as a function of emission wavelengths (400-560 nm) shows at least three components. The f of the slow component increases from 400 to 440 nm, then decreases, whereas the f of the intermediate and fast components behave in the opposite way. Results indicate that: (i) the fast component is associated with the emission at 530 nm; (ii) the slow component is associated with the emission at 420 nm; (iii) a fast additive component, characterized by a very short lifetime, is present on the blue side of the emission spectrum; (iv) the intermediate component results from overlapping contributions, including the emission of the band at 490 nm, that could not be resolved; (v) the increased emission at 490 nm, caused by acetate binding, is likely due to the stabilization of the ketoenamine tautomer induced by an increase in polarity of the active site microenvironment and/or a decrease in proton dissociation in the excited state; (vi) excitation at 330 nm, where the enolimine tautomer absorbs, leads to emission decays typical of the ketoenamine.

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.

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.

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.

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.

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.

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.

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.

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.

Kinetic isotope effects as a probe of the beta-elimination reaction catalyzed by O-acetylserine sulfhydrylase.

Primary and alpha-secondary deuterium kinetic isotope effects have been measured for the O-acetylserine sulfhydrylase from Salmonella typhimurium using both steady-state and single-wavelength stopped-flow studies. Data suggest an asymmetric transition state for alpha-proton abstraction by the active site lysine and the elimination of the acetyl group of O-acetyl-L-serine (OAS) to form the alpha-aminoacrylate intermediate. The value of D(V/KOAS) using OAS-2-d is dependent on pH from 5.8 to 7.0 with independent values of 2.8 and 1.7 estimated at low and high pH, respectively. Thus, OAS is sticky, and a value of 1.5 is calculated for the forward commitment to catalysis, indicating that the OAS external Schiff base preferentially partitions toward the alpha-aminoacrylate intermediate compared to OAS being released from enzyme. The intrinsic primary deuterium isotope effect determined from single-wavelength stopped-flow studies of alpha-proton abstraction by the active site lysine is about 2.0. D(V/KOAS) and T(V/KOAS) were determined as 2.6 +/- 0.1 and 4.2 +/- 0.2 at pH 6.1, respectively, giving a calculated intrinsic deuterium isotope effect of 3.3 +/- 0.9, consistent with the D(V/KOAS) obtained from steady-state studies at low pH. The alpha-secondary deuterium kinetic isotope effect using OAS-3,3-d2 is 1.11 +/- 0.06 obtained by direct comparison of initial velocities and 1.2 obtained by single-wavelength stopped-flow experiments. Data can be compared to a value of 1.81 +/- 0.04 using OAS-3,3-d2 for alpha-DKeq for the first half-reaction.

Isotope partitioning with Ascaris suum phosphofructokinase is consistent with an ordered kinetic mechanism.

Isotope partitioning and initial velocity studies have been used to study the kinetic mechanism of Ascaris suum phosphofructokinase (PFK) at pH 8.0 for the native enzyme (nPFK), and at pH 6.8 for a form of enzyme desensitized (dPFK) to hysteresis in the reaction time course, to ATP allosteric inhibition, and to F6P homotropic cooperativity. Complete trapping (P*max approximately equal to 100%) of the E:MgATP* complex as fructose (1-32P)-1, 6-bisophosphate for both enzyme forms is consistent with the previously proposed steady-state ordered mechanism [Rao, G.S.J., Harris, B.G., & Cook, P.F. (1987) J.Biol. Chem. 262, 14074-14079] with MgATP binding before fructose 6-phosphate (F6P). K'F6P values for trapping of MgATP of 0.54 +/- 0.09 mM for nPFK and 0.85 +/- 0.15 mM for dPFK were obtained. Saturating amounts of the heterotropic activator fructose 2, 6-bisphosphate (F26P2) gives no change in the trapping parameters for nPFK with a P*max of 100% and a K'F6P of 0.40 +/- 0.06 mM. For dPFK, however, F26P2 causes a decrease in both parameters, giving a P*max of 54% and a K'F6P of 0.26 +/- 0.07 mM. The partial trapping of E:MgATP* in the presence of F26P2 for dPFK suggests that the activator changes the kinetic mechanism from an ordered to a random binding of substrates. Initial velocity studies confirm the change in mechanism. Uncompetitive inhibition by arabinose 5-phosphate (Ara5P), a dead-end inhibitory analog of F6P, versus MgATP for nPFK in the absence and presence of F26P2 is consistent with an ordered mechanism with MgATP adding to enzyme prior to F6P. An uncompetitive pattern is also obtained with dPFK for Ara5P versus MgATP in the absence of F26P2, but the pattern becomes noncompetitive in the presence of F26P2, consistent with a change to a random mechanism. No trapping of the E:[14C]F6P complex could be detected, indicating either that the E:[14C]F6P complex does not form in a significant amount under the conditions used or that the off-rate for F6P from enzyme is much faster than the net rate constant for formation of the first product, FBP. The data are consistent with a predominantly ordered mechanism with MgATP binding prior to F6P. The minor pathway with MgATP dissociating from the E:F6P:MgATP ternary complex becomes apparent for the dPFK in the presence of F26P2.

Formation of the alpha-aminoacrylate immediate limits the overall reaction catalyzed by O-acetylserine sulfhydrylase.

O-Acetylserine sulfhydrylase-A (OASS-A) catalyzes the final step in the synthesis of L-cysteine, viz., the beta-substitution of acetate in O-acetyl-L-serine (OAS) by sulfide via a ping-pong kinetic mechanism . Rapid-scanning stopped-flow and single-wavelength absorbance and fluorescence stopped-flow experiments were carried out to obtain information on the location and amount of limitation of rate-determining steps for the overall reaction and the individual half-reactions of OASS-A. The first half-reaction, conversion of OAS to the alpha-aminoacrylate intermediate and acetate, is rate-limiting for the overall reaction catalyzed by OASS-A. No intermeidates are detected within the second half-reaction, and thus rate constants for all steps must be > or = 1000s-1 at the lowest sulfide concentration used. Within the first half reaction, formation of the extrernal Schiff base (Kassociation = 0.2 mM-1) is observed in the first milliseconds, followed by its slower conversion to the alpha-aminocacrylate intermediate with a rate constant of 300 s-1, close to the value of 130 s-1 obtained for V/Et [Tai, C.H., Nalabolu, S.R., Jacobson, T.M., Minter D.E., & Cook, P.F. (1993) Biochemistry 32, 6433-6442]. Addition of L-cysterine ot OASS-A results in a rapid formation of the external Schiff base (Kassociation = 6.7 mM-1), followed by transient formation of the alpha-aminoacylate intermediate with a slightly lower rate (70-100 s-1) compared to OAS. The alpha-aminoacrylate intermediate decays to generate a species absorbing maximally at 418 nm, resulting from attack of the cysteine thiol to give ether in external Schiff base linkage with the active site PLP.

Comparison of the substrate specificities of cAMP-dependent protein kinase from bovine heart and Ascaris suum muscle.

The catalytic subunits of cAMP-dependent protein kinases (protein kinase A) from bovine heart and Ascaris suum muscle exhibit only 48% sequence identity and show quantitative differences in substrate specificity. These differences were not obvious at the level of short synthetic substrate peptides but were distinct for some protein substrates. Phosphofructokinase from Ascaris, a physiological substrate, was a better substrate for the protein kinase from the nematode in comparison to the mammalian protein kinase due to a 10-fold lower Michaelis constant. Selective phosphorylation by the two kinases was also observed with some in vitro substrates. In addition, quantitative differences in the interactions between R- and C-subunits from Ascaris and bovine heart were observed. However, several synthetic peptides whose sequence reflected the phosphorylation site of Ascaris suum phosphofructokinase (AKGRSDS*IV), or variations of it, were phosphorylated with the same efficiency by both protein kinases. Based on the data the following are concluded: (1) In agreement with the conservation of structure in the catalytic cleft, the recognition of substrates by protein kinases from phylogenetically distant organisms exhibits similarity. (2) Non-conserved parts of the surface of the protein kinase molecule may contribute to binding of protein substrates and thus to selective recognition.