Multicomponent telerehabilitation programme for older veterans with multimorbidity: a programme evaluation.

INTRODUCTION: Older veterans with multimorbidity experience physical, mental and social factors which may negatively impact health and healthcare access. Physical function, behaviour change skills and loneliness may not be addressed during traditional physical rehabilitation. Thus, a multicomponent telerehabilitation programme could address these unmet needs. This programme evaluation assessed the safety, feasibility and change in patient outcomes for a multicomponent telerehabilitation programme. METHODS: Individuals were eligible if they were a veteran/spouse, age ≥50 years and had ≥3 comorbidities. The telerehabilitation programme included four core components: (1) High-intensity rehabilitation, (2) Coaching interventions, (3) Social support and (4) Technology. Physical therapists delivered the 12-week programme and collected patient outcomes at baseline, 4 weeks, 8 weeks and 12 weeks. Programme evaluation measures included safety events (occurrence and type), feasibility (adherence) and patient outcomes (physical function). Safety and feasibility outcomes were analysed using descriptive statistics. The mean pre-post programme difference and 95% CI for patient outcomes were generated using paired t-tests. RESULTS: Twenty-one participants enrolled in the telerehabilitation programme; most were male (81%), white (72%) and non-Hispanic (76%), with an average of 5.7 (3.0) comorbidities. Prevalence of insession safety events was 3.2% (0.03 events/session). Fifteen (71.4%) participants adhered to the programme (attended ≥80% of sessions). Mean (95% CI) improvements for physical function are as follows: 4.7 (2.4 to 7.0) repetitions for 30 s sit to stand, 6.0 (4.0 to 9.0) and 5.0 (2.0 to 9.0) repetitions for right arm curl and left arm curl, respectively, and 31.8 (15.9 to 47.7) repetitions for the 2 min step test. CONCLUSION: The telerehabilitation programme was safe, feasible and demonstrated preprogramme to postprogramme improvements in physical function measures while addressing unmet needs in a vulnerable population. These results support a randomised clinical trial while informing programme and process adaptations.

Using biological-physical modelling for informing sea lice dispersal in Loch Linnhe, Scotland.

Sea lice are a constraint on the sustainable growth of Scottish marine salmonid aquaculture. As part of an integrated pest management approach, farms coordinate procedures within spatial units. We present observations of copepodids being at relatively greater density than nauplii in upper waters, which informs the development of surface layer sea lice transmission modelling of Loch Linnhe, Scotland, for informing farm parasite management. A hydrodynamic model is coupled with a biological particle-tracking model, with characteristics of plankton sea lice. Simulations are undertaken for May and October 2011-2013, forced by local wind data collected for those periods. Particles are continually released from positions representing farm locations, weighted by relative farm counts, over a 2-week period and tracked for a further 5 days. A comparison is made between modelled relative concentrations against physical and biological surveys to provide confidence in model outputs. Connectivity between farm locations is determined in order to propose potential coordination areas. Generally, connectivity depends on flow patterns in the loch and decreases with increased farm separation. The connectivity indices are used to estimate the origins of the sea lice population composition at each site, which may influence medicinal regimens to avoid loss of efficacy.

Determining the age of individual Lepeophtheirus salmonis (Krøyer, 1837) copepodids by measuring stored lipid volume; proof of principle.

Confocal microscopy has facilitated measurement of stained lipid volume in Lepeophtheirus salmonis copepodid larvae. Quantity of lipid, location and morphology of vesicles may allow an estimate of age and viability.

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.

Initial velocity, spectral, and pH studies of the serine-glyoxylate aminotransferase from Hyphomicrobiuim methylovorum.

Serine-glyoxylate aminotransferase (SGAT) from Hyphomicrobium methylovorum is a pyridoxal 5'-phosphate (PLP) enzyme that catalyzes the interconversion of L-serine and glyoxylate to hydroxypyruvate and glycine. The initial velocity and dead-end inhibition patterns are consistent with a ping-pong kinetic mechanism. The Km values for L-serine and the alternative substrate ketomalonate are 0.28 +/- 0.02 and 1.13 +/- 0.08 mM, respectively. The spectrum of SGAT at pH 7.5 shows an absorbance maximum at 413 nm and a shoulder centered at 330 nm corresponding to the ketoenamine and enolimine forms of the protonated Schiff's base with the enolimine tautomer predominating. As determined by the changes in the enzyme absorbance spectrum the enzyme can be converted from the E-PLP to the E-pyridoxamine 5'-phosphate (E-PMP) form on addition of L-serine. The enzyme can subsequently be converted back to E-PLP by addition of glyoxylate or hydroxypyruvate. The enzyme displays a pH-dependent spectral change with a pK of about 8.2 which is ascribed to the ionization of an enzymatic residue that effects the tautomeric equilibrium between the ketoenamine and enolimine tautomers of the protonated aldimine. The V/K(L-serine) pH profile displays two pK values at pH 7.5 and 8.5 with limiting slopes of 1 and -1. The V/K(ketomalonate) pH profile displays one pK at 8.2 on the basic side with a limiting slope of 1 and the log K(I oxalate) pH profile shows one pK on the basic side at pH 7.2. The data suggest the active enzyme is the protonated aldimine and an enzymatic base with a pK of 7.5 accepts a proton from the alpha-amine of substrate to initiate catalysis.

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.

Glutamate 325 is a general acid-base catalyst in the reaction catalyzed by fructose-2,6-bisphosphatase.

A bifunctional enzyme, fructose-6-phosphate, 2-kinase:fructose-2, 6-bisphosphatase, catalyzes synthesis and hydrolysis of fructose 2, 6-bisphosphate. The phosphatase reaction occurs in two steps: the formation of a phosphoenzyme intermediate and release of beta-D-fructose 6-phosphate, followed by hydrolysis of the phosphoenzyme. The objective of this study was to determine whether E325 in the Fru 2,6-Pase active site is an acid-base catalyst. The pH-rate profile for k(cat) for the wild-type enzyme exhibits pK values of 5.6 and 9.1. The pH dependence of k(cat) for the E325A mutant enzyme gives an increase in the acidic pK from 5.6 to 6.1. Formate, acetate, propionate, and azide accelerate the rate of hydrolysis of the E325A mutant enzyme, but not of the wild-type enzyme. Azide and formate, the smallest of the weak acids tested, are the most potent activators. The k(cat) vs pH profile of the E325A mutant enzyme in the presence of formate is similar to that of the wild-type enzyme. Taken together, these data are consistent with E325 serving an acid-base role in the phosphatase reaction. The exogenous low MW weak acids act as a replacement general base in the hydrolysis of the phosphoenzyme intermediate, rescuing some of the activity lost upon eliminating the glutamate side chain.

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.

Lysine 199 is the general acid in the NAD-malic enzyme reaction.

Site-directed mutagenesis was used to change K199 in the Ascaris suum NAD-malic enzyme to A and R and Y126 to F. The K199A mutant enzyme gives a 10(5)-fold decrease in V and a 10(6)-fold decrease in V/K(malate) compared to the WT enzyme. In addition, the ratio for partitioning of the oxalacetate intermediate toward pyruvate and malate changes from a value of 0.4 for the WT enzyme to 1.6 for K199A, and repeating the experiment with A-side NADD gives isotope effects of 3 and 1 for the WT and K199A mutant enzymes, respectively. The K199R mutant enzyme gives only a factor of 10 decrease in V, and the pK for the general acid in this mutant enzyme has increased from 9 for the WT enzyme to >10 for the K199R mutant enzyme. Tritium exchange from solvent into pyruvate is catalyzed by the WT enzyme, but not by the K199A mutant enzyme. The Y126F mutant enzyme gives a 10(3)-fold decrease in V. The oxalacetate partition ratio and isotope effect on oxalacetate reduction for the Y126F mutant enzyme are identical, within error, to those measured for the WT enzyme. Thus, Y126 is important to the overall reaction, but its role at present is unclear. Data are consistent with K199 functioning as the general acid that protonates C3 of enolpyruvate to generate the pyruvate product in the malic enzyme reaction.

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.

Expression and stereochemical and isotope effect studies of active 4-oxalocrotonate decarboxylase.

4-Oxalocrotonate decarboxylase (4-OD) and vinylpyruvate hydratase (VPH) from Pseudomonas putida mt-2 form a complex that converts 2-oxo-3-hexenedioate to 2-oxo-4-hydroxypentanoate in the catechol meta fission pathway. To facilitate mechanistic and structural studies of the complex, the two enzymes have been coexpressed and the complex has been purified to homogeneity. In addition, Glu-106, a potential catalytic residue in VPH, has been changed to glutamine, and the resulting E106QVPH mutant has been coexpressed with 4-OD and purified to homogeneity. The 4-OD/E106QVPH complex retains full decarboxylase activity, with comparable kinetic parameters to those observed for 4-OD in the wild-type complex, but is devoid of any detectable hydratase activity. Decarboxylation of (5S)-2-oxo-3-[5-D]hexenedioate by either the 4-OD/VPH complex or the mutant complex generates 2-hydroxy-2,4E-[5-D]pentadienoate in D(2)O. Ketonization of 2-hydroxy-2,4-pentadienoate by the wild-type complex is highly stereoselective and results in the formation of 2-oxo-(3S)-[3-D]-4-pentenoate, while the mutant complex generates a racemic mixture. These results indicate that 2-hydroxy-2, 4-pentadienoate is the product of 4-OD and that 2-oxo-4-pentenoate results from a VPH-catalyzed process. On this basis, the previously proposed hypothesis for the conversion of 2-oxo-3-hexenedioate to 2-oxo-4-hydroxypentanoate has been revised [Lian, H., and Whitman, C. P. (1994) J. Am. Chem. Soc. 116, 10403-10411]. Finally, the observed (13)C kinetic isotope effect on the decarboxylation of 2-oxo-3-hexenedioate by the 4-OD/VPH complex suggests that the decarboxylation step is nearly rate-limiting. Because the value is not sensitive to either magnesium or manganese, it is likely that the transition state for carbon-carbon bond cleavage is late and that the metal positions the substrate and polarizes the carbonyl group, analogous to its role in oxalacetate decarboxylase.

Substitution of pyridoxal 5'-phosphate in D-serine dehydratase from Escherichia coli by cofactor analogues provides information on cofactor binding and catalysis.

D-Serine dehydratase (DSD) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the conversion of D-serine to pyruvate and ammonia. Spectral studies of enzyme species where the natural cofactor was substituted by pyridoxal 5'-sulfate (PLS), pyridoxal 5-deoxymethylene phosphonate (PDMP), and pyridoxal 5'-phosphate monomethyl ester (PLPMe) were used to gain insight into the structural basis for binding of cofactor and substrate analogues. PDMP-DSD exhibits 35% of the activity of the native enzyme, whereas PLS-DSD and PLPMe-DSD are catalytically inactive. The emission spectrum of native DSD when excited at 280 nm shows maxima at 335 and 530 nm. The energy transfer band at 530 nm is very likely generated as a result of the proximity of Trp-197 to the protonated internal Schiff base. The cofactor analogue-reconstituted DSD species exhibit emission intensities decreasing from PLS-DSD, to PLPMe-DSD, and PDMP-DSD, when excited at 415 nm. Large increases in fluorescence intensity at 530 (540) nm can be observed for cofactor analogue-reconstituted DSD in the presence of substrate analogues when excited at 415 nm. In the absence and presence of substrate analogues, virtually identical far UV CD spectra were obtained for all DSD species. The visible CD spectra of native DSD, PDMP-DSD, and PLS-DSD exhibit a band centered on the visible absorption maximum with nearly identical intensity. Addition of substrate analogues to native and cofactor analogue-reconstituted DSD species results in most cases in a decrease or elimination of ellipticity. The results are interpreted in terms of local conformational changes and/or changes in the orientation of the bound cofactor (analogue).

Lysine 183 is the general base in the 6-phosphogluconate dehydrogenase-catalyzed reaction.

Site-directed mutagenesis was used to change K183 of sheep liver 6-phosphogluconate dehydrogenase to A, E, H, C, Q, R, and M to probe its possible role as a general base catalyst. Each of the mutant proteins was characterized with respect to its kinetic parameters at pH 7 and the pH dependence of kinetic parameters for the K183R mutant enzyme. The only mutant enzyme that gives a significant amount of catalysis is the K183R mutant, and the extent of catalysis is decreased by about 3 orders of magnitude; the general base pK is perturbed to a pH value of >9. All other mutant enzymes exhibit rates that are decreased by about 4 orders of magnitude compared to that of the wild-type enzyme. Data are consistent with the general base function of K183.

Mapping the active site topography of the NAD-malic enzyme via alanine-scanning site-directed mutagenesis.

The NAD-malic enzyme cDNA has been subcloned into the pQE expression vector, expressed with a six-His tag, and purified. The His-tagged enzyme is purified by a combination of Ni-NTA and orange A agarose column chromatography with a yield of 45% and an estimated purity of >90%. The tag and linker have no effect on the kinetic parameters of the enzyme compared to the wild-type enzyme. Alanine-scanning site-directed mutagenesis has been carried out on all of the conserved neutral acid residues of the NAD-malic enzyme from Ascaris suum. Data obtained confirm the predicted role of D178 and D295 in metal ion binding, the likely role of D294, D361, and E440 in the NAD binding site, and the role of E58 and D272 in malate binding. Decreases in V/E(t) by 10(4)-fold and in V/K(malate)E(t) by 10(7)-fold, when D295 is changed to alanine, suggest that it is a likely candidate for the general base that accepts a proton from the malate hydroxyl in the oxidation step.

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.

Alpha-secondary tritium kinetic isotope effects indicate hydrogen tunneling and coupled motion occur in the oxidation of L-malate by NAD-malic enzyme.

The NAD-malic enzyme from Ascaris suum catalyzes the divalent metal ion-dependent oxidative decarboxylation of L-malate to give pyruvate and CO2, with NAD+ as the oxidant. Alpha-secondary tritium kinetic isotope effects were measured with NAD+ or APAD+ and L-malate-2-H(D) and several different divalent metal ions. The alpha-secondary tritium kinetic isotope effects are slightly higher than 1 with NAD+ and L-malate as substrates, much larger than the expected inverse isotope effect for a hybridization change from sp2 to sp3. The alpha-secondary tritium kinetic isotope effects are reduced to values near 1 with L-malate-2-D as the substrate, regardless of the metal ion that is used. Data suggest the presence of quantum mechanical tunneling and coupled motion in the malic enzyme reaction when NAD+ and malate are used as substrates. Isotope effects were also measured using the D/T method with NAD+ and Mn2+ as the substrate pair. A Swain-Schaad exponent of 2.2 (less than the value of 3.26 expected for strictly semiclassical behavior) is estimated, suggesting the presence of other slow steps along the reaction pathway. With APAD+ and Mn2+ as the substrate pair, inverse alpha-secondary tritium kinetic isotope effects are observed, and a Swain-Schaad exponent of 3.3 is estimated, consistent with rate-limiting hydride transfer and no quantum mechanical tunneling or coupled motion. Data are discussed in terms of the malic enzyme mechanism and the theory developed by Huskey for D/T isotope effects as an indicator of tunneling [Huskey, W. P. (1991) J. Phys. Org. Chem. 4, 361-366].

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.

Reaction mechanism of fructose-2,6-bisphosphatase. A mutation of nucleophilic catalyst, histidine 256, induces an alteration in the reaction pathway.

A bifunctional enzyme, fructose-6-phosphate,2-kinase/fructose 2, 6-bisphosphatase (Fru-6-P,2-kinase/Fru-2,6-Pase), catalyzes synthesis and degradation of fructose 2,6-bisphosphate (Fru-2,6-P2). Previously, the rat liver Fru-2,6-Pase reaction (Fru-2,6-P2 --> Fru-6-P + Pi) has been shown to proceed via a phosphoenzyme intermediate with His258 phosphorylated, and mutation of the histidine to alanine resulted in complete loss of activity (Tauler, A., Lin, K., and Pilkis, S. J. (1990) J. Biol. Chem. 265, 15617-15622). In the present study, it is shown that mutation of the corresponding histidine (His256) of the rat testis enzyme decreases activity by less than a factor of 10 with a kcat of 17% compared with the wild type enzyme. Mutation of His390 (in close proximity to His256) to Ala results in a kcat of 12.5% compared with the wild type enzyme. Attempts to detect a phosphohistidine intermediate with the H256A mutant enzyme were unsuccessful, but the phosphoenzyme is detected in the wild type, H390A, R255A, R305S, and E325A mutant enzymes. Data demonstrate that the mutation of His256 induces a change in the phosphatase hydrolytic reaction mechanism. Elimination of the nucleophilic catalyst, H256A, results in a change in mechanism. In the H256A mutant enzyme, His390 likely acts as a general base to activate water for direct hydrolysis of the 2-phosphate of Fru-2,6-P2. Mutation of Arg255 and Arg305 suggests that the arginines probably have a role in neutralizing excess charge on the 2-phosphate and polarizing the phosphoryl for subsequent transfer to either His256 or water. The role of Glu325 is less certain, but it may serve as a general acid, protonating the leaving 2-hydroxyl of Fru-2,6-P2.