Biomedical subjects

P F Cook

Publications and source records attributed to P F Cook.

At least 55 records · Page 3Linked to original sources

PubMed1996

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.

Catalysis↗

PubMed1996

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.

Adenosine Triphosphate↗

PubMed1996

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.

Alanine↗

PubMed1996

Role of the divalent metal ion in the NAD:malic enzyme reaction: an ESEEM determination of the ground state conformation of malate in the E:Mn:malate complex.

The conformation of L-malate bound at the active site of Ascaris suum malic enzyme has been investigated by electron spin echo envelope modulation spectroscopy. Dipolar interactions between Mn2+ bound to the enzyme active site and deuterium specifically placed at the 2-position, the 3R-position, and the 3S-position of L-malate were observed. The intensities of these interactions are related to the distance between each deuterium and Mn2+. Several models of possible Mn-malate complexes were constructed using molecular graphics techniques, and conformational searches were conducted to identify conformers of malate that meet the distance criteria defined by the spectroscopic measurements. These searches suggest that L-malate binds to the enzyme active site in the trans conformation, which would be expected to be the most stable conformer in solution, not in the gauche conformer, which would be more similar to the conformation required for oxidative decarboxylation of oxalacetate formed from L-malate at the active site of the enzyme.

Animals↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed1995

Resolution of pyridoxal 5'-phosphate from O-acetylserine sulfhydrylase from Salmonella typhimurium and reconstitution of apoenzyme with cofactor and cofactor analogues as a probe of the cofactor binding site.

A procedure has been developed to prepare the apoenzyme of O-acetylserine sulfhydrylase (apoOASS) by first converting the native enzyme to the alpha-aminoacrylate intermediate and dialyzing against 5 M guanidinium chloride. Aposulfhydrylase is stable for at least a month in buffers containing phosphate or phosphate analogues. Reconstitution of aposulfhydrylase with pyridoxal 5'-phosphate (PLP), 2'-methyl PLP (2'-MePLP), and pyridoxal 5'-deoxymethylenephosphonate (PDMP) results in enzymatically competent proteins. Pyridoxal in the absence and presence of phosphate and pyridoxal 5'-phosphate monomethyl ester are unable to form a Schiff base with apoOASS. The reconstitution of apoOASS with PLP is highly cooperative judged by the initial rate of activity regained and shows no evidence of saturation with PLP. The reconstituted enzymes have been studied using 31P NMR spectroscopy. The 31P NMR of the aposulhydrylase reconstituted with PLP exhibits a chemical shift of 5.2 ppm, identical to that of native enzyme. The latter has been interpreted in terms of a strong ionic interaction between enzyme and the 5'-phosphate of PLP (P. F. Cook, S. Hara, S. Nalabolu, and K. D. Schnackerz, 1992, Biochemistry 31, 2298-2303). Reconstitution with 2'-MePLP gives a lower chemical shift of 4.95 ppm, suggesting a weaker ionic interaction at the 5'-phosphate when compared to native enzyme. The PDMP-reconstituted enzyme gives a chemical shift of 23.7 ppm, consistent with the monoanionic form of the bound phosphonate. All of the chemical shifts are pH independent. The apoenzyme has also been reconstituted with pyridoxal 5'-sulfate. Although the resulting enzyme is not active in the overall reaction, it forms the external Schiff base. The PDMP- and 2'-MePLP-reconstituted enzymes have also been studied in the presence of amino acid reactants and analogues, and results are discussed in terms of the mechanism of OASS.

Apoenzymes↗

PubMed1995

Equilibrium model in an in vitro poly(ADP-ribose) turnover system.

Poly(ADP-ribose) metabolism plays an important role in numerous DNA-related functions. This homopolymer is synthesized by poly(ADP-ribose) polymerase and is degraded mainly by the poly(ADP-ribose) glycohydrolase. The activities of these two enzymes in the nucleus are closely coordinated. To better understand the interactions between these enzymes, we designed an in vitro system in which both enzymes are present at the same time. In this work, we report a model describing the synthesis and degradation of the poly(ADP-ribose) in turnover conditions. Because the half-life of the polymer in the cell is close to 1 min, we studied the very early kinetic interactions of these two enzymes.

Adenosine Diphosphate Ribose↗

PubMed1995

A pH-dependent allosteric transition in Ascaris suum phosphofructokinase distinct from that observed with fructose 2,6-bisphosphate.

Ascaris suum phosphofructokinase exhibits dramatic shifts in its circular dichroic spectra in the pH range 6 to 8. These shifts are quite distinct from those induced by the activators AMP and fructose 2,6-bisphosphate. Concomitant with these pH-induced spectral shifts, the enzyme also displays changes in its allosteric behavior. Inorganic ions such as K+, NH+4, SO4(2-), and PO4(3-) also cause CD-spectral shifts similar to those produced by a change in pH. Based on the evidence derived from gel filtration and sedimentation equilibrium studies, the observed CD-spectral shifts are interpreted as due to conformational changes in the enzyme tetramer rather than due to a change in its aggregation state. Further, since the pK value of 6.4 obtained from pH dependence of increase in ellipticity at 210 nm agrees very well with the pK value of 6.8 for the loss of ATP inhibition due to modification of a histidine residue (G. S. J. Rao, B. A. Wariso, P. F. Cook, and B. G. Harris (1987) J. Biol. Chem. 262, 14068-14073), it is concluded that a single histidine residue in the ATP-inhibitory site acts as a trigger for the structural changes accompanying ATP inhibition of the enzyme. This view is strongly supported by the observation that the enzyme desensitized to ATP inhibition by chemical modification of a histidine residue in the ATP-inhibitory site shows absolutely no change in its CD spectrum in the pH range 6 to 8. This study demonstrates that the mechanism of activation of phosphofructokinase at higher pH and by inorganic ions involves conformational transitions that are quite distinct from those induced by AMP and fructose 2,6-bisphosphate. A scheme is presented that incorporates all of the different states of the enzyme dependent upon effectors and pH.

Adenosine Monophosphate↗

PubMed1995

Identification and spectral characterization of the external aldimine of the O-acetylserine sulfhydrylase reaction.

The O-acetylserine sulfhydrylase (OASS) reaction has been studied using a number of spectral probes including UV--visible, fluorescence, circular dichroism, and 31P NMR spectroscopy. The addition of L-cysteine, L-alanine, and glycine to OASS results in a shift in lambda max of 412 nm for the internal Schiff base to 418 nm resulting from the formation of the external Schiff base. The addition of L-serine or O-methyl-D,L-serine gives decreases of the absorbance of unliganded enzyme at 412 nm of about 50% and 20%, respectively, concomitant with an increase in the absorbance at 320 nm and a shift in the lambda max of the remaining visible absorbance to 418 nm. The spectral shifts observed in the presence of L-serine are suggestive of establishing an equilibrium between different forms of external Schiff base. The concentration dependence of the changes at 440 (L-cysteine) and 320 nm (L-serine) provides an estimate of the dissociation constant for the external aldimine. The pH dependence of the dissociation constant suggests the alpha-amine of the amino acid must be unprotonated for nucleophilic attack at C4' of PLP, and an enzyme side chain must be unprotonated to hydrogen-bond the thiol or hydroxyl side chain of the amino acid. When L-cysteine is the amino acid, the thiol side chain must be protonated to hydrogen-bond to the unprotonated enzyme side chain. The 31P NMR chemical shift is increased from 5.2 ppm for unliganded enzyme to 5.3 ppm in the presence of L-cysteine, signaling a tighter interaction at the 5'-phosphate upon formation of the external Schiff base.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1995

Acid-base chemical mechanism of O-acetylserine sulfhydrylases-A and -B from pH studies.

The pH dependence of kinetic parameters using natural and alternative reactants was determined in order to obtain information on the chemical mechanisms of the A and B isozymes of O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium. A general mechanism is proposed for OASS in which OAS binds with its alpha-amine unprotonated to carry out a nucleophilic attack on C4' of the protonated Schiff base and with the acetyl carbonyl hydrogen-bonded to a protonated enzyme group (or a water molecule), which aids in the beta-elimination of acetate. The enzyme lysine that was in Schiff base linkage with the active site pyridoxal 5'-phosphate deprotonates the alpha-carbon in the beta-elimination reaction, and a proton is likely released with the acetate product. Sulfide likely binds as HS- to undergo nucleophilic attack on the alpha-aminoacrylate intermediate, followed by protonation of the alpha-carbon by the enzyme lysine. In OASS-A, HS- is hydrogen-bonded to the enzyme group that assists in the beta-elimination of acetate, but this is not the case for OASS-B. The pH independent equilibrium constant for the first half-reaction of OASS-A is 1.6 x 10(-3), while the second half-reaction is practically irreversible.

Acetates↗

PubMed1995

Acid-base chemical mechanism of aspartase from Hafnia alvei.

An acid-base chemical mechanism is proposed for Hafnia alvei aspartase in which a proton is abstracted from C-3 of the monoanionic form of L-aspartate by an enzyme general base with a pK of 6.3-6.6 in the absence and presence of Mg2+. The resulting carbanion is presumably stabilized by delocalization of electrons into the beta-carboxyl with the assistance of a protonated enzyme group in the vicinity of the beta-carboxyl. Ammonia is then expelled with the assistance of a general acid group that traps an initially expelled NH3 as the final NH4+ product. In agreement with the function of the general acid group, potassium, an analog of NH4+, binds optimally when the group is unprotonated. The pK for the general acid is about 7 in the absence of Mg2+, but is increased by about a pH unit in the presence of Mg2+. Since the same pK values are observed in the pKi(succinate) and V/K pH profile, both enzyme groups must be in their optimum protonation state for efficient binding of reactant in the presence of Mg2+. At the end of a catalytic cycle, both the general base and general acid groups are in a protonation state opposite that in which they started when aspartate was bound. The presence of Mg2+ causes a pH-dependent activation of aspartase exhibited as a partial change in the V and V/Kasp pH profiles. When the aspartase reaction is run in D2O to greater than 50% completion no deuterium is found in the remaining aspartate, indicating that the site is inaccessible to solvent during the catalytic cycle.

Acid-Base Equilibrium↗

PubMed1995

Acid-base catalytic mechanism and pH dependence of fructose 2,6-bisphosphate activation of the Ascaris suum phosphofructokinase.

A form of phosphofructokinase (PFK) from Ascaris suum desensitized to hysteresis in the reaction time course and ATP allosteric inhibition has been used to study the activation by fructose 2,6-bisphosphate (F26P2) at varied pH in both reaction directions. In the direction of phosphorylation of F6P, V and V/KMgATP are constant over the pH range 6-9, while V/KF6P decreases at low pH, giving a pK value of 7.0, and at high pH, giving a pK of 8.9. V and V/KMgATP are insensitive to the presence of F26P2, but V/KF6P is increased by a constant amount in the presence of saturating F26P2 over the entire pH range studied. The concentration of F26P2 that gives half the change in V/KF6P, Kact, increases as the pH decreases, giving a pK of 7.4, reflecting an enzyme group that must be unprotonated for optimum binding of F26P2. In the direction of phosphorylation of MgADP, V and V/KMgADP are pH-independent, and both are insensitive to the presence of F26P2. V/KFBP decreases at high pH, giving a pK of about 7.3, and is increased by a constant amount in the presence of F26P2 over the entire pH range studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1995

Metal ion activator effects on intrinsic isotope effects for hydride transfer from decarboxylation in the reaction catalyzed by the NAD-malic enzyme from Ascaris suum.

The mechanism of the oxidative decarboxylation reaction catalyzed by the NAD-malic enzyme from Ascaris suum has been examined with several different divalent metal ion activators and dinucleotide substrates. Primary deuterium and tritium isotope effects have been obtained and, in combination with the partitioning ratios of the oxalacetate intermediate to malate and pyruvate, have been used to calculate commitment factors, intrinsic deuterium isotope effects on the hydride transfer step, and intrinsic 13C isotope effects for the decarboxylation step. A survey of malate analogs has been undertaken to define the geometry of the active site and to identify functional groups on malate important for substrate binding. With NAD as dinucleotide substrate, a direct correlation between the size of the divalent metal ion activator and the intrinsic deuterium isotope effect is observed. An isotope effect significantly greater than the semiclassical limit is seen when Cd2+ is the metal ion activator, indicating a substantial tunneling contribution. The primary intrinsic 13C isotope effect on the decarboxylation step increases over the series Mg2+ < Mn2+ < Cd2+, which is in contrast to the equal isotope effects measured for these metal ions for the nonenzymatic decarboxylation of oxalacetate [Grissom, C. B., & Cleland, W. W. (1986) J. Am. Chem. Soc. 108, 5582]. With Mn2+ or Cd2+ as the divalent metal ion activator, the data support a stepwise mechanism for the enzymatic oxidative decarboxylation with NAD as the dinucleotide substrate, but a change to a concerted mechanism is indicated with more redox-positive dinucleotide substrates as suggested previously with Mg2+ as activator [Karsten, W. E., & Cook, P. F. (1994) Biochemistry 33, 2096].(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Kinetic mechanism of serine transacetylase from Salmonella typhimurium.

Serine transacetylase from Salmonella typhimurium was purified to near homogeneity. A complete initial velocity study in both reaction directions suggests that the enzyme catalyzes the conversion of acetyl CoA and L-serine to O-acetyl-L-serine (OAS) and coenzyme A (CoASH) by a ping pong bi bi kinetic mechanism. Initial velocity patterns in the absence of added inhibitors in both directions are best described by a series of parallel lines. Product inhibition by OAS is competitive with respect to acetyl CoA and noncompetitive with respect to L-serine, while product inhibition by L-serine is competitive against CoASH and noncompetitive against OAS. Glycine and S-methyl-L-cysteine (SMC) were used as dead-end analogs of L-serine and OAS, respectively. Glycine is competitive against L-serine, and uncompetitive against acetyl CoA, while SMC is competitive against OAS and uncompetitive against CoASH. All of the above inhibition patterns are consistent with those predicted for a single site ping pong bi bi kinetic mechanism. The equilibrium constant for the transacetylase reaction is 15 in the direction of serine acetylation. The constant was measured by monitoring the change in CoASH concentration obtained for reactions in which the ratio of acetyl CoA/CoASH was constant and the ratio of OAS/serine was varied. The Keq calculated from the Haldane relationship is in good agreement with the value obtained by direct measurement.

Acetyl Coenzyme A↗

PubMed1994

Stepwise versus concerted oxidative decarboxylation catalyzed by malic enzyme: a reinvestigation.

The NAD-malic enzyme catalyzes the divalent metal-ion-dependent oxidative decarboxylation of L-malate to yield CO2, pyruvate, and the reduced dinucleotide. With Mg2+ as the divalent metal ion activator, primary deuterium and tritium isotope effects have been obtained with several different alternative dinucleotide substrates. The partitioning ratio of oxalacetate to malate and pyruvate has also been determined with either NAD or 3-acetylpyridine adenine dinucleotide (3-APAD). These data have been used to calculate estimates of commitment factors and intrinsic isotope effects for the NAD-malic enzyme reaction. The calculated values of the intrinsic 13C and deuterium isotope effects with NADP are similar to the previously determined values for the chicken liver malic enzyme (Grissom, C.B., & Cleland, W. W. (1988) Biochemistry 27, 2927) and suggest that the transition-state structures are similar for the Ascaris NAD- and chicken liver NADP-malic enzymes. With NAD or NADP as the dinucleotide substrate, the data are all consistent with a stepwise chemical mechanism with oxidation of L-malate at C2 preceding decarboxylation of the bound oxalacetate intermediate. However, none of the data with the alternative dinucleotide substrates, 3-acetylpyridine adenine dinucleotide and 3-pyridine aldehyde adenine dinucleotide (PAAD), can be fit with satisfaction to the various criteria that support a stepwise mechanism with NAD(P). The mechanism with 3-APAD and PAAD is likely concerted. The most likely explanation for a change in the mechanism for oxidative decarboxylation from stepwise with NAD(P) to concerted with alternative dinucleotide substrates such as 3-APAD and PAAD is a difference in the configuration of bound malate when the different dinucleotide substrates are used.

Animals↗

PubMed1994

Lanthanide pyrophosphates as substrates for the pyrophosphate-dependent phosphofructokinases from Propionibacterium freudenreichii and Phaseolus aureus: evidence for a second metal ion required for reaction.

In the absence of Mg2+, both the dimeric bacterial and tetrameric plant fructose 2,6-bisphosphate-activated pyrophosphate-dependent phosphofructokinases (PPi-PFKs) are inactive at pH 8 and 25 degrees C. In the presence of a low concentration of Mg2+ (5 microM), both enzymes will utilize a variety of metal-pyrophosphate complexes as reactant in the direction of fructose 6-phosphate (F6P) phosphorylation. The Vmax values are about 100-fold lower and the Km values about 10-fold greater than those measured with MgPPi when lanthanide-PPi complexes are used as a substrate. In the presence of added Mg2+, the Km values of the above remain essentially unchanged, while Vmax values increase 10-fold for lanthanide-PPi complexes. These data, along with the 12-16 order of magnitude increased affinity of the lanthanides for PPi compared to Mg2+, indicate that the PPi-PFKs require two metal ions for catalysis, one to form a chelate with PPi and a second as an essential activator. With CePPi, an activation constant of about 25 microM is measured for Mg2+. In addition, a number of other divalent (but no tripositive) metal ions serve as activators including Mn2+, Co2+, Mo2+, Cr2+, Fe2+, and Ni2+; activation constants are in the range 20-150 microM. The exchange-inert CrIII(PPi)(H2O)4 complex is not a substrate, but is an inhibitor competitive against MgPPi with a Ki of 27 microM. Results are discussed in terms of the possible role of the divalent metal ion activators.

Binding Sites↗

PubMed1994

Product binding to the alpha-carboxyl subsite results in a conformational change at the active site of O-acetylserine sulfhydrylase-A: evidence from fluorescence spectroscopy.

The intrinsic fluorescence of the pyridoxal 5'-phosphate (PLP) enzyme O-acetylserine sulfhydrylase-A (OASS-A) was studied in order to gain insight into the structural basis for binding of substrates and products and for catalysis. Excitation of OASS-A with 298-nm light gives an emission spectrum with two maxima, 337 and 498 nm. OASS-A has two tryptophan residues, and the 337-nm maximum indicates that at least one of these is exposed somewhat to aqueous solvent. The 498-nm emission observed is due to fluorescence of the PLP Schiff base. Some of this long-wavelength fluorescence is likely due to direct excitation by incident radiation. However, the concomitant quenching of 340-nm emission and the enhancement of 498-nm emission observed upon reconstitution of apoenzyme with PLP support the conclusion that some of the long-wavelength emission is due to singlet-singlet transfer from at least one tryptophan residue to the PLP Schiff base. Enhancement of 498-nm fluorescence by either of the products, acetate or cysteine, of the enzymatic reaction without a quenching of 337-nm fluorescence is consistent with triplet-singlet transfer from one or both of the tryptophan residues to the PLP Schiff base. This would require a rigid environment for the tryptophan donor when the product is bound. However, a conformational change which affected principally the environment of the PLP Schiff base, resulting in a longer lifetime of its excited singlet state, would also increase the intensity of the 498-nm emission. Enhancement of OASS-A long-wavelength fluorescence by each product requires the unprotonated form of a different group on enzyme. Enhancement by acetate binding requires the unprotonated form of an enzyme group with a pK of 7 and is insensitive to substitution on the methyl group. L-Cysteine binding enhances 498-nm fluorescence when a group with a pK of 8 is unprotonated, and substitution at the thiol or the methylene bridge does not affect the enhancement elicited. Binding of L-cysteine to free enzyme (E) likely results in the formation of the external Schiff base accompanied by a conformational change giving fluorescence enhancement. The carboxylate moiety of acetate likely binds to the alpha-carboxylate subsite for amino acid reactants such as L-cysteine, resulting in a conformational change in the internal Schiff base and giving rise to the observed fluorescence enhancement. Data are interpreted in terms of the mechanism of OASS-A.

Acetates↗

PubMed1993

pH dependence of the kinetic mechanism of the adenosine 3',5'-monophosphate dependent protein kinase catalytic subunit in the direction of magnesium adenosine 5'-diphosphate phosphorylation.

In order to determine the acid-base chemical mechanism of the adenosine 3',5'-monophosphate dependent protein kinase catalytic subunit, the pH dependence of the kinetic mechanism in the direction of MgADP phosphorylation has been determined using initial velocity studies in the presence and absence of dead-end inhibitors. The kinetic mechanism in the direction of MgADP phosphorylation is random, with changes in the preference of substrate binding as a function of pH. At pH 7.2 and below, the kinetic mechanism is ordered with phosphorylated peptide binding prior to MgADP. At pH 7.6, the opposite pathway with MgADP binding prior to phosphorylated peptide is preferred. The pH independence of V/Et is consistent with a mechanism in which reactants only bind to the correctly protonated form of the enzyme. The V/KMgADP value decreases, as the pH increases, giving a pK of about 7 which is likely that of a general acid, the same group that serves as a general base in the opposite reaction direction [Yoon, M.-Y., & Cook, P.F. (1987) Biochemistry 26, 4118]. The pKiPSP decreases at low pH giving a pK of about 6.5, probably reflecting the phosphate group of the peptide.

Adenosine Diphosphate↗