A copper-methionine interaction controls the pH-dependent activation of peptidylglycine monooxygenase

The pH dependence of native peptidylglycine monooxygenase (PHM) and its M314H variant has been studied in detail. For wild-type (WT) PHM, the intensity of the Cu-S interaction visible in the Cu(I) extended X-ray absorption fine structure (EXAFS) data is inversely proportional to catalytic activity over the pH range of 3-8. A previous model based on more limited data was interpreted in terms of two protein conformations involving an inactive Met-on form and an active flexible Met-off form [Bauman, A. T., et al. (2006) Biochemistry 45, 11140-11150] that derived its catalytic activity from the ability to couple into vibrational modes critical for proton tunneling. The new studies comparing the WT and M314H variant have led to the evolution of this model, in which the Met-on form has been found to be derived from coordination of an additional Met residue, rather than a more rigid conformer of M314 as previously proposed. The catalytic activity of the mutant decreased by 96% because of effects on both k(cat) and K(M), but it displayed the same activity-pH profile with a maximum around pH 6. At pH 8, the reduced Cu(I) form gave spectra that could be simulated by replacement of the Cu(M) Cu-S(Met) interaction with a Cu-N/O interaction, but the data did not unambiguously assign the ligand to the imidazole side chain of H314. At pH 3.5, the EXAFS still showed the presence of a strong Cu-S interaction, establishing that the Met-on form observed at low pH in WT cannot be due to a strengthening of the Cu(M)-methionine interaction but must arise from a different Cu-S interaction. Therefore, lowering the pH causes a conformational change at one of the Cu centers that brings a new S donor residue into a favorable orientation for coordination to copper and generates an inactive form. Cys coordination is unlikely because all Cys residues in PHM are engaged in disulfide cross-links. Sequence comparison with the PHM homologues tyramine β-monooxygenase and dopamine β-monooxygenase suggests that M109 (adjacent to H site ligands H107 and H108) is the most likely candidate. A model is presented in which H108 is protonated with a pK(a) of 4.6 to generate the inactive low-pH form with Cu(H) coordinated by M109, H107, and H172.     

Analysis of the cold lability behavior of rabbit muscle phosphofructokinase.

Rabbit skeletal muscle phosphofructokinase has been previously shown to exhibit the characteristic of cold lability in phosphate buffers at pH values below pH 7 [Bock, P.E. and Frieden, C. (1974) Biochemistry $\text{13}$, 4191–4196]. Studies of the residual activity as a function of pH, reflecting the equilibrium between active and inactive forms of the enzyme, have been performed. These experiments show that the cold lability can be ascribed to a shift of the apparent pK describing the pH-dependent inactivation to lower pH values at higher temperatures. The apparent pK, Hill interaction coefficient and heat of ionization for this process indicate that the equilibrium between the inactive and active forms of the enzyme may be controlled by the ionization of one or more histidine residues per enzyme subunit. In addition the apparent pK for the pH dependence of the residual activity at constant temperature is influenced by the presence of ligands which are substrates or effectors of the phosphofructokinase reaction.     

Phosphofructokinase. I. Mechanism of the pH-dependent inactivation and reactivation of the rabbit muscle enzyme.

The kinetics of inactivation and reactivation of rabbit skeletal muscle phosphofructokinase have been studied as a function of pH and enzyme concentration at constant temperature in phosphate buffer. From the enzyme concentration dependence, we conclude that the minimal mechanism for inactivation involves a protonation step followed by isomerization to an inactive form and then dissociation to a species of one-half the molecular weight. Other data indicate a subsequent isomerization of the dissociated form. The pH and temperature dependence of the inactivation process shows that it is controlled by ionizable groups, and that the apparent pK for these groups is temperature-dependent in such a way as to make the enzyme show the characteristic of cold lability below pH 7. Reactivation of the inactive enzyme occurs by a kinetically different pathway involving deprotonation of an inactive, dissociated form to a form which may either isomerize to another inactive form, or dimerize to the active enzyme. A general mechanism is postulated in which the inactivation and reactivation processes are different aspects of the same mechanism. This mechanism assumes four species (two containing four subunits and two containing two subunits) each of which can exist in a protonated and unprotonated form. Inactivation or reactivation induced by changes in pH or temperature reflect the kinetic establishment of a new steady state between these forms. How the apparent pK values which control the distribution of the enzyme between protonated and unprotonated forms describe the pH-dependent characteristics of the enzyme is discussed in terms of the proposed mechanism.     

Phosphoglycolate phosphatase. Effect of cation and pH on activity.

P-glycolate phosphatase requires divalent cations for activity. Activity-pH curves identified 2 active site residues with pK values at pH 5.7 and pH 9.1 in the presence of magnesium and at pH 5.7 and pH 7.5 in the presence of manganese or cobalt. Saturation velocity kinetics enabled the identification of two distinct divalent cation binding sites. The first, nonspecific site has a K0.5 of 2 to 7 x 10(-5) M, depending on the cation and the pH. The second site, which is specific for magnesium, binds this cation in a negatively cooperative fashion. The affinity at pH 8.1 varies approximately 100-fold from the first magnesium bound to the fourth. The negative cooperativity is greatest at high pH. Because the pH range of activity is very broad, both the phosphate monoanion and dianion of P-glycolate must be bound as the substrate. The concentration of these two species at the apparent Km is independent of magnesium concentration. The P-glycolate.magnesium complex is kinetically inactive.     

Effects of strong electrolyte upon the activity of Clostridium perfringens sialidase toward sialyllactose and sialoglycolipids.

Clostridium perfringens sialidase was purified by affinity chromatography. Kinetic properties of the enzyme were examined with sialyllactose and with mixed sialoglycolipids (gangliosides) as substrates. With the latter substrate in 0.01 M Tris-acete in the absence of strong electrolyte, the pH optimum for enzymatic activity was 6.8. Addition of strong electrolyte (0.01 to 0.10 M Nac1) to the reaction medium caused an acidic shift and a broadening of the pH optimum, Enzymatic activity at pH 5.8 rose approximately 2.5-fold; a concomitant loss of activity at pH 6.8 was also observed. The alteration of enzymatic activity caused by strong electrolyte were dependent upon changes in Vmax. Km remained nearly invariant. Thus, a reversible transition of the enzyme from a relatively inactive to a highly active form occurred as a function of strong electrolyte concentration. Determination of the pK values of the active functional groups of C. perfringens sialidase revealed that the effects of strong electrolyte were exerted upon the pKa group of the enzyme. Strong electrolyte appeared to shield unfavorable electrostatic interactions between polyanionic sialoglycolipid micelles and the enzyme molecule, thus protecting the pKa group from inactivation. In comparision with the effects of strong electrolyte upon enzymatic activity toward the sialoglycolipid substrate, those observed with the monovalent substrate, sialyllacthose, were minor. Collectively, these findings indicate that ionic environment may effectively control the activity and relative substrate specificity of C. perfringens sialidase at a given pH. Furthermore, they explain the low pH optima and skewed pH profiles previously reported for enzymatic activity toward high molecular weight substrates.     

Stopped-flow kinetic studies of the cellulase-catalyzed conversion of cellulose to glucose

The kinetics of the cellulase-catalyzed conversion of soluble cellulose into glucose have been studied over a range of substrate concentrations and temperatures, and at pH values ranging from 4.75 to 7.0. Lineweaver-Burk plots were linear and led to V = 6.2muM/s and K(m) = 13.1 mM at pH 5.8 and 25.0 degrees C. The pK values corresponding to the free enzyme are 4.8 and 6.8 and are consistent with carboxyl and imidazole groups as the active ionizing species. These pK values were little changed in the enzyme-substrate intermediate that reacts in the ratedetermining step, suggesting that the ionizing groups are still free in this intermediate. The activation energy corresponding to V/K(m) is 80.6 kJ/mol, and that corresponding to V is 38.7 kJ/mol. The corresponding entropies of activation are 21 J K(-1) mol(-1) and -157 J K(-1) mol(-1), respectively.     

Studies of glutamate dehydrogenase. Regulation of glutamate dehydrogenase from Candida utilis by a pH and temperature-dependent conformational transition.

Glutamate dehydrogenase from Candida utilis undergoes a reversible conformational transition between an active and an inactive state at low pH AND low temperature. This conformational transition can also be followed by fluorescence measurements. The temperature-dependent equilibrium between the active and the inactive state is characterized by a transition temperature of 10.7 degrees C and a delta H value of 148 kcal/mol (620 kJ/mol). The temperature dependence of the enzymic activity above 15 degrees C yields an activation energy of 15 kcal/mol (63 kJ/mol), a larger value than that for the beef liver enzyme (9 kcal/mol; 38 kJ/mol). In contrast to the yeast enzyme the Arrhenius plot is linear and, therefore, the beef liver enzyme is not transformed into an inactive conformation at low temperatures. Sedimentation analysis shows that the inactivation of the Candida utilis enzyme is not caused by change in the quaternary structure. The pH dependence of the conformational transition at low pH measured by fluorescence change is characterized by a pK value of 7.01 for the enzyme in the absence and of 6.89 for the enzyme in the presence of 2-oxoglutarate with a Hill coefficient of 3.4 in both cases. Similar results are found when the pH dependence of the enzymic activity is analyzed. With the beef liver enzyme the same pK value is obtained but with a Hill coefficient of 1 indicating cooperativity only in the case of the Candida utilis enzyme. The best fit of the pH dependence of the rate constants of the fluorescence changes was obtained with pK values of 7.45 and 6.45 for the active and the inactive state respectively. In this model the lowest time constant which is obtained at the pH of the equilibrium was found to be 0.05 s-1. Preincubation experiments with the substrate 2-oxoglutarate but not with the coenzyme shift the equilibrium to the active conformation. The coenzyme obviously reduces the rate constant of the conformational transition. The sedimentation coefficient (SO20, w) and the molecular weight were found to be 11.0 S and 276 000, respectively. The enzyme molecule is built up by six polypeptide chains each having a molecular weight of 47 000.     

Moving a phenol hydroxyl group from the surface to the interior of a protein: effects on the phenol potential and pK(A)

De novo protein design and electrochemistry were used to measure changes in the potential and pK(A) of a phenol when its OH group is moved from a solvent-exposed to a sequestered protein position. A "phenol rotation strategy" was adopted in which phenols, containing a SH in position 4, 3, or 2 relative to the OH group, were bound to a buried protein site. The alpha(3)C protein used here is a tryptophan to cysteine variant of the structurally defined alpha(3)W protein (Dai et al. (2002) J. Am. Chem. Soc. 124, 10952-10953). The protein characteristics of alpha(3)C and the three mercaptophenol-alpha(3)C (MP-alpha(3)C) proteins are shown to be close to those of alpha(3)W. Moreover, the phenol OH group is fully solvent exposed in 4MP-alpha(3)C and more sequestered in 3MP-alpha(3)C and 2MP-alpha(3)C. Here we compare the redox properties of the three mercaptophenols when bound to alpha(3)C and to cysteine free in water. The pK(A) and E(peak) values are essential identical when 4MP is ligated to alpha(3)C relative to when it is free in solution. In contrast, these values are increased in 3MP-alpha(3)C and 2MP-alpha(3)C relative to the solvated compounds. The E(peak) vs pH plots all display a approximately 59 mV/pH unit dependence. We conclude that interactions with the OH group dominate the phenol redox characteristics. In 3MP-alpha(3)C and 2MP-alpha(3)C, hydrogen bonds between the protein and the bound phenols appear to either stabilize the reduced phenol or destabilize the radical, relative to the aqueous buffer, raising the potential by 0.11 and 0.12 V, respectively.     

Enzyme deactivation due to metal-ion dissociation during turnover of the cobalt-beta-lactamase catalyzed hydrolysis of beta-lactams

Metallo-beta-lactamases are native zinc enzymes that catalyze the hydrolysis of beta-lactam antibiotics but are also able to function with cobalt (II) and require one or two metal ions for catalytic activity. The kinetics of the hydrolysis of benzylpenicillin catalyzed by cobalt substituted beta-lactamase from Bacillus cereus (BcII) are biphasic. The dependence of enzyme activity on pH and metal-ion concentration indicates that only the di-cobalt enzyme is catalytically active. A mono-cobalt enzyme species is formed during the catalytic cycle, which is virtually inactive and requires the association of another cobalt ion for turnover. Two intermediates with different metal to enzyme stoichiometries are formed on a branched reaction pathway. The di-cobalt enzyme intermediate is responsible for the direct catalytic route, which is pH-independent between 5.5 and 9.5 but is also able to slowly lose one bound cobalt ion via the branching route to give the mono-cobalt inactive enzyme intermediate. This inactivation pathway of metal-ion dissociation occurs by both an acid catalyzed and a pH-independent reaction, which is dependent on the presence of an enzyme residue of pK(a) = 8.9 +/- 0.1 in its protonated form and shows a large kinetic solvent isotope effect (H(2)O/D(2)O) of 5.2 +/- 0.5, indicative of a rate-limiting proton transfer. The pseudo first-order rate constant to regenerate the di-cobalt beta-lactamase from the mono-cobalt enzyme intermediate has a first-order dependence on cobalt-ion concentration in the pH range 5.5-9.5. The second-order rate constant for metal-ion association is dependent on two groups of pK(a) 6.32 +/- 0.1 and 7.47 +/- 0.1 being in their deprotonated basic forms and one group of pK(a) 9.48 +/- 0.1 being in its protonated form.     

APS kinase from Penicillium chrysogenum. Dissociation and reassociation of subunits as the basis of the reversible heat inactivation

Adenosine-5'-phosphosulfate (APS) kinase from Penicillium chrysogenum, loses catalytic activity at temperatures greater than approximately 40 degrees C. When the heat-inactivated enzyme is cooled to 30 degrees C or lower, activity is regained in a time-dependent process. At an intermediary temperature (e.g. 36 degrees C) an equilibrium between active and inactive forms can be demonstrated. APS kinase from P. chrysogenum is a dimer (Mr = 57,000-60,000) composed of two apparently identical subunits. Three lines of evidence suggest that the reversible inactivation is a result of subunit dissociation and reassociation. (a) Inactivation is a first-order process. The half-time for inactivation at a given temperature is independent of the original enzyme concentration. Reactivation follows second-order kinetics. The half-time for reactivation is inversely proportional to the original enzyme concentration. (b) The equilibrium active/inactive ratio at 36 degrees C increases as the total initial enzyme concentration is increased. However, Keq,app at 5 mM MgATP and 36 degrees C calculated as [inactive sites]2/0.5 [active sites] is near-constant at about 1.7 X 10(-8) M over a 10-fold concentration range of enzyme. (c) At 46 degrees C, the inactive P. chrysogenum enzyme (assayed after reactivation) elutes from a calibrated gel filtration column at a position corresponding to Mr = 33,000. Substrates and products of the APS kinase reaction had no detectable effect on the rate of inactivation. However, MgATP and MgADP markedly stimulated the reactivation process (kapp = 3 X 10(5) M-1 X s-1 at 30 degrees C and 10 mM MgATP). The kapp for reactivation was a nearly linear function of MgATP up to about 20 mM suggesting that the monomer has a very low affinity for the nucleotide compared to that of the native dimer. Keq,app at 36 degrees C increases as the MgATP concentration is increased. The inactivation rate constant increased as the pH was decreased but no pK alpha could be determined. The reactivation rate constant increased as the pH was increased. An apparent pK alpha of 6.4 was estimated.     

Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions

The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82 ppm and 182.87 ppm on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5 ppm to 182.9 ppm on binding to thermolysin. From pH studies we conclude that the pK(a) of the inhibitor carboxylate group is lowered by at least 1.5 pK(a) units when it binds to either enzyme. The signal at ~183 ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of the carboxylate carbon of Z-[1-(13)C]-L-tryptophan is ≤3.71? from the active site cobalt atom of thermolysin. We conclude that the side chain of Z-[1-(13)C]-L-tryptophan is not bound in the S(2)' subsite of thermolysin. As the chemical shifts of the carboxylate carbons of the bound inhibitors are all ~183 ppm we conclude that they are all bound in a similar way most probably with the inhibitor carboxylate group directly coordinated to the active site zinc atom. Our spectrophotometric results confirm that the active site zinc atom is tetrahedrally coordinated when the inhibitors Z-tryptophan or Z-phenylalanine are bound to thermolysin.     

Mechanism of chalcone synthase. pKa of the catalytic cysteine and the role of the conserved histidine in a plant polyketide synthase

Polyketide synthases (PKS) assemble structurally diverse natural products using a common mechanistic strategy that relies on a cysteine residue to anchor the polyketide during a series of decarboxylative condensation reactions that build the final reaction product. Crystallographic and functional studies of chalcone synthase (CHS), a plant-specific PKS, indicate that a cysteine-histidine pair (Cys(164)-His(303)) forms part of the catalytic machinery. Thiol-specific inactivation and the pH dependence of the malonyl-CoA decarboxylation reaction were used to evaluate the potential interaction between these two residues. Inactivation of CHS by iodoacetamide and iodoacetic acid targets Cys(164) in a pH-dependent manner (pK(a) = 5.50). The acidic pK(a) of Cys(164) suggests that an ionic interaction with His(303) stabilizes the thiolate anion. Consistent with this assertion, substitution of a glutamine for His(303) maintains catalytic activity but shifts the pK(a) of the thiol to 6.61. Although the H303A mutant was catalytically inactive, the pH-dependent incorporation of [(14)C]iodoacetamide into this mutant exhibits a pK(a) = 7.62. Subsequent analysis of the pH dependence of the malonyl-CoA decarboxylation reaction catalyzed by wild-type CHS and the H303Q and C164A mutants also supports the presence of an ion pair at the CHS active site. Structural and sequence conservation of a cysteine-histidine pair in the active sites of other PKS implies that a thiolate-imidazolium ion pair plays a central role in polyketide biosynthesis.     

Investigations of the alkaline and acid transitions of umecyanin, a stellacyanin from horseradish roots

The effect of pH on Cu(I) and Cu(II) umecyanin (UCu), a phytocyanin obtained from horseradish roots, has been studied by electronic and NMR spectroscopy and using direct electrochemical measurements. A pK(a) value of approximately 9.5-9.8 is observed for the alkaline transition in UCu(II), and this leads to a slightly altered active site structure, as indicated by the changes in the paramagnetic 1H NMR spectrum. Electrochemical studies show that the pK(a) value for this transition in UCu(I) is 9.9. The alkaline transition is caused by the deprotonation of a surface lysine residue, with Lys96 being the most likely candidate. The isotropically shifted resonances in the (1)H NMR spectrum of UCu(II) also shift upon lowering the pH (pK(a) 5.8), and this can be assigned to the protonation of the surface (noncoordinating) His65 residue. This histidine titrates in UCu(I) with a pK(a) of 6.3. The reduction potential of the protein in this range is also dependent on pH, and pK(a) values matching those from NMR, for the two oxidation states of the protein, are obtained. There is no evidence for either of the active site histidines (His44 and His90) titrating in UCu(I) in the pH range studied (down to pH 3.7). Also highlighted in these studies are the remarkable active site similarities between umecyanin and the other phytocyanins which possess an axial Gln ligand.     

On the activation of bovine chymotrypsinogen A. Preparation of alanine-neochymotrypsinogen and its activation to alpha-chymotrypsin

Alanine-neochymotrypsinogen was prepared by incubating 20 parts bovine pancreas chymotrypsinogen A with one part alpha-chymotrypsin in a solution containing 1 M (NH4)2SO4, 0.1 M sodium acetate, 0.05 M Tris buffer (pH 8.0) and 0.5 mg/ml soybean trypsin inhibitor. Optimal yields of NH2-terminal alanine were obtained after 60 h incubation at 4 degrees C. Ala-neochymotrypsinogen was isolated from the reaction mixture by affinity chromatography and ion-exchange chromatography on carboxymethyl-cellulose. As expected, the purified preparation was enzymatically inactive and, compared to chymotrypsinogen, had one additional NH2-terminal group identified as alanine. Ala-neochymotrypsinogen was activated by incubating with trypsin at a zymogen : trypsin ratio of 30 : 1 in 0.1 M phosphate buffer, pH 7.6 at 4 degrees C for 1 h. The fully active, stable species was identified as alpha-chymotrypsin.     

Exploring the function of Tyr83 in bacteriorhodopsin: features of the Y83F and Y83N mutants.

Tyrosine-83, a residue which is conserved in all halobacterial retinal proteins, is located at the extracellular side in helix C of bacteriorhodopsin. Structural studies indicate that its hydroxyl group is hydrogen bonded to Trp189 and possibly to Glu194, a residue which is part of the proton release complex (PRC) in bacteriorhodopsin. To elucidate the role of Tyr83 in proton transport, we studied the Y83F and Y83N mutants. The Y83F mutation causes an 11 nm blue shift of the absorption spectrum and decreases the size of the absorption changes seen upon dark adaptation. The light-induced fast proton release, which accompanies formation of the M intermediate, is observed only at pH above 7 in Y83F. The pK(a) of the PRC in M is elevated in Y83F to about 7.3 (compared to 5.8 in WT). The rate of the recovery of the initial state (the rate of the O --> BR transition) and light-induced proton release at pH below 7 is very slow in Y83F (ca. 30 ms at pH 6). The amount of the O intermediate is decreased in Y83F despite the longer lifetime of O. The Y83N mutant shows a similar phenotype in respect to proton release. As in Y83F, the recovery of the initial state is slowed several fold in Y83N. The O intermediate is not seen in this mutant. The data indicate that the PRC is functional in Y83F and Y83N but its pK(a) in M is increased by about 1.5 pK units compared to the WT. This suggests that Tyr83 is not the main source for the proton released upon M formation in the WT; however, Tyr83 is involved in the proton release affecting the pK(a) of the PRC in M and the rate of proton transport from Asp85 to PRC during the O --> bR transition. Both the Y83F and the Y83N mutations lead to a greatly decreased functionality of the pigment at high pH because most of the pigment is converted into the inactive P480 species, with a pK(a) 8-9.     

Focusing of pepsin in strongly acidic immobilized pH gradients

A new acrylamido buffer has been synthesized, for use in isoelectric focusing in immobilized pH gradients. This compound (2-acrylamido glycolic acid) has a pK = 3.1 (at 25 degrees C, 20 mM concentration during titration) and is used, by titration with the pK 9.3 Immobiline, to produce a linear pH gradient in the pH 2.5-3.5 interval. Pepsin (from pig stomach) focused in this acidic pH gradient is resolved into four components, two major (with pI values 2.76 and 2.78) and two minor (having pI values 2.89 and 2.90). This is the first time that such strongly acidic proteins could be focused in an immobilized pH gradient. Even in conventional isoelectric focusing in amphoteric buffers it has been impossible to focus reproducibly very-low-pI macromolecules.     

Elucidation of isoform-dependent pH sensitivity of troponin i by NMR spectroscopy

Myocardial ischemia is characterized by reduced blood flow to cardiomyocytes, which can lead to acidosis. Acidosis decreases the calcium sensitivity and contractile efficiency of cardiac muscle. By contrast, skeletal and neonatal muscles are much less sensitive to changes in pH. The pH sensitivity of cardiac muscle can be reduced by replacing cardiac troponin I with its skeletal or neonatal counterparts. The isoform-specific response of troponin I is dictated by a single histidine, which is replaced by an alanine in cardiac troponin I. The decreased pH sensitivity may stem from the protonation of this histidine at low pH, which would promote the formation of electrostatic interactions with negatively charged residues on troponin C. In this study, we measured acid dissociation constants of glutamate residues on troponin C and of histidine on skeletal troponin I (His-130). The results indicate that Glu-19 comes in close contact with an ionizable group that has a pK(a) of ～6.7 when it is in complex with skeletal troponin I but not when it is bound to cardiac troponin I. The pK(a) of Glu-19 is decreased when troponin C is bound to skeletal troponin I and the pK(a) of His-130 is shifted upward. These results strongly suggest that these residues form an electrostatic interaction. Furthermore, we found that skeletal troponin I bound to troponin C tighter at pH 6.1 than at pH 7.5. The data presented here provide insights into the molecular mechanism for the pH sensitivity of different muscle types.     

Reactions of dysprosium with adenine nucleotides

Dysprosium catalyzes a rapid hydrolysis of both ATP and ADP, at ambient temperatures, pH 7.0, where no hydroxide precipitates. The reactive complexes, at pH 6.7, were found to contain 2Dy:1ATP and 3Dy:2ADP. AMP forms an insoluble complex containing 1Dy:2AMP, which does not hydrolyze. ATP also forms a soluble 1Dy:1ATP complex, which does not react. Dysprosium only catalyzes the hydrolysis of ATP above pH 5.8, where it has been titrated to the hydroxide. At the optimum pH (pH 7) the stoichiometric composition is Dy2.ATP.(OH)2, indicating that the active complex is neutral, whereas at pH 5.8 the stoichiometric composition is Dy2.ATP.(OH)+, indicating an inactive cationic complex. The mechanism proposed for the hydrolysis is consistent with those proposed for other in vitro systems known to catalyze the hydrolysis of ATP.     

pK(a) calculations for class C beta-lactamases: the role of Tyr-150

The Poisson-Boltzmann method was used to compute the pK(a) values of titratable residues in a set of class C beta-lactamases. In these calculations, the pK(a) of the phenolic group of residue Tyr150 is the only one to stand out with an abnormally low value of 8.3, more than one pK(a) unit lower than the measured reference value for tyrosine in solution. Other important residues of the catalytic pocket, such as the conserved Lys67, Lys315, His314, and Glu272 (hydrogen-bonded to the ammonium group of Lys315), display normal protonation states at neutral pH. pK(a) values were also computed in catalytically impaired beta-lactamase mutants. Comparisons between the relative k(cat) values and the Tyr150 pK(a) value in these mutants revealed a striking correlation. In active enzymes, this pK(a) value is always lower than the solution reference value while it is close to normal in inactive enzymes. These results thus support the hypothesis that the phenolate form of Tyr150 is responsible for the activation of the nucleophilic serine. The possible roles of Lys67 and Lys315 during catalysis are also discussed.     

Yeast hexokinase A. Succinylation and properties of the active subunit.

Yeast hexokinase A (ATP:D-hexose 6-phosphotransferase, EC2.7.1.1) dissociates into its subunits upon reaction with succinic anhydride. The chemically modified subunits could be isolated in a catalytically active form. The Km values found for ATP and for glucose were of the some order as those found for the native enzyme. Of the 37 amino groups present per enzyme subunit, 2-3 of these groups might be located in the proximity of the region of subunit interactions. The 50% loss of the initial activity, which follows the succinylation of these more reactive amino groups, does not seem to be due to the modification of a residue on the enzyme active site or to a change of the tertiary structure of the protein. This 50%loss of the enzyme activity may be related to the dissociation of the dimer into monomers. Both native enzyme and the succinylated subunits have the same H-dependent denaturation rate profiles in response to 2 M urea. Moreover, the apparent pK of the group involved in the transition from a more stable conformation of the protein in the acid range to a less stable one at alkaline pH seems to be similar to the pK of the group implicated in the transition between the protonated inactive form of the enzyme and an active deprotonated form. The succinylated subunit presents 'negative co-operativity' with respect to ATP at slightly acid pH; however, the burst-type slow transient in the reaction progress curve and the activation effect induced by physiological polyanions, effects observed for the native enzyme, were not detected in the standard experimental conditions with the succinylated subunit.