Competitive, reversible inhibition of cytosolic phospholipase A2 at the lipid-water interface by choline derivatives that partially partition into the phospholipid bilayer.

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. When assaying the human recombinant cPLA2 using membranes isolated from [3H]arachidonate-labeled U937 cells as substrate, 2-(2'-benzyl-4-chlorophenoxy)ethyl-dimethyl-n-octadecyl-ammonium chloride (compound 1) was found to inhibit the enzyme in a dose-dependent manner (IC50 = 5 microM). It was over 70 times more selective for the cPLA2 as compared with the human nonpancreatic secreted phospholipase A2, and it did not inhibit other phospholipases. Additionally, it inhibited arachidonate production in N-formyl-methionyl-leucyl-phenylalanine-stimulated U937 cells. To further characterize the mechanism of inhibition, an assay in which the enzyme is bound to vesicles of 1,2-dimyristoyl-sn -glycero-3-phosphomethanol containing 6-10 mol % of 1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine was employed. With this substrate system, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the lipid-water interface. The apparent equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface (KI*app) was determined to be 0.097 +/- 0.032 mol % versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.3 +/- 0.1 mol %. Thus, compound 1 represents a novel structural class of inhibitor of cPLA2 that partitions into the phospholipid bilayer and competes with the phospholipid substrate for the active site. Shorter n-alkyl-chained (C-4, C-6, C-8) derivatives of compound 1 were shown to have even smaller KI*app values. However, these short-chained analogs were less potent in terms of bulk inhibitor concentration needed for inhibition when using the [3H]arachidonate-labeled U937 membranes as substrate. This discrepancy was reconciled by showing that these shorter-chained analogs did not partition into the [3H]arachidonate-labeled U937 membranes as effectively as compound 1. The implications for in vivo efficacy that result from these findings are discussed.

Evaluation of the kinetic mechanism of Escherichia coli uridine diphosphate-N-acetylmuramate:L-alanine ligase.

Initial velocity methods were used to probe the kinetic mechanism of Escherichia coli uridine diphosphate-N-acetylmuramate:L-alanine ligase (UNAM:L-Ala ligase). When the activity (in the forward direction) versus substrate concentration data were plotted in double-reciprocal form, all line patterns were intersecting. The best fit of these data was to the equation for an ordered mechanism with the following parameters: k(cat), 1000 +/- 100 min(-1); Kma, 210 +/- 40 microM; Kmb, 84 +/- 20 microM; Kmc, 70 +/- 15 microM; Kia, 180 +/- 50 microM; Kib, 68 +/- 24 microM. Initial velocity line patterns were also determined when the concentration of one substrate was varied at different fixed concentrations of a second substrate while the third substrate was held at a concentration more than 100 times its Km value. Reciprocal plots of data collected with either ATP or L-alanine present at more than 100 times their Km values resulted in intersecting line patterns. Data collected with UNAM present at 100 times its Km value gave a set of parallel lines. These data are consistent with UNAM binding as the second substrate in an ordered mechanism. ADP, uridine diphosphate-N-acetylmuramoyl-L-alanine (UNAMA), and phosphate were tested as product inhibitors versus substrates. None of the products were competitive inhibitors versus L-alanine or UNAM, while the only observed competitive inhibition was ADP versus ATP. These results are consistent with an ordered kinetic mechanism wherein ATP binds first, UNAM binds second, and ADP is the last product released. Rapid quench experiments were performed in the presence of all three substrates or in the presence of ATP and UNAM. The production of acid-labile phosphate as a function of time is characterized by a burst phase followed by a slower linear phase with the rate close to k(cat) in the presence of all three substrates. Only a burst phase was observed for the time course of the reaction in the presence of ATP and UNAM. In both cases, the burst rate was identical. These observations are consistent with L-alanine being the third substrate to bind in a sequential mechanism involving a putative acyl-phosphate intermediate.

Presence of glycerol masks the effects of phosphorylation on the catalytic efficiency of cytosolic phospholipase A2.

Cytosolic phospholipase A2 catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. The enzymatic activity of cPLA2 is affected by several mechanisms, including substrate presentation and the phosphorylation state of the enzyme. Using covesicles of 1-palmitoy1-2-arachidonoyl-[arachidonoyl-1-14C]-8n-glycero-3 -phosphocholine and 1,2-dimyristoyl-phosphatidylmethanol as substrate, the effects of phosphorylation on the interfacial binding and catalytic constants were investigated. Phosphorylated and dephosphorylated enzyme forms were shown to have identical values of 2.6 microM for KMapp, an equilibrium dissociation constant which consists of the intrinsic dissociation constant from the lipid/water interface (Ks) and the dissociation constant for phospholipid from the active site (KM*). Moreover, the values of KM* for phosphorylated and dephosphorylated enzyme did not differ significantly (0.4 +/- 0.1 and 0.2 +/- 0.1, respectively). However, dephosphorylation of the enzyme reduced the value of kcat by 39%. The phosphorylation state of the enzyme had no effect on either the cooperativity shown by this enzyme or the thermal stability of the enzyme. Surprisingly, the presence of glycerol (4 M) masks the effect of phosphorylation on kcat. Instead, glycerol increased the value of kcat by 440% for the phosphorylated enzyme and by 760% for the dephosphorylated form. Moreover, addition of glycerol had only small effects on KMapp. the increase in the kcat upon addition of glycerol results from a substantial decrease in the activation energy from 29.4 to 14.8 kcal. mol-1. To determine whether the effects of phosphorylation of the enzyme or addition of glycerol are unique to this artificial substrate, membranes from U937 cells were isolated and used as substrate. With these membranes, the dephosphorylated enzyme was only 21% less active than the phosphorylated enzyme. In the presence of glycerol, there was no detectable difference the two enzyme forms, and the rate of hydrolysis was increased by 300-390% over that measured in the absence of glycerol. These results suggest that the catalytic efficiency of the phosphorylated enzyme is not particularly relevant to its activation in vivo. Moreover, it may be that glycerol is mimicking the effect of some unidentified factor which greatly enhances the catalytic efficiency of the enzyme.

Spectroscopic properties of Escherichia coli UDP-N-acetylenolpyruvylglucosamine reductase.

Purified uridine diphosphate N-acetylenolpyruvylglucosamine reductase (E.C. 1.1.1.158) was analyzed by circular dichroism (CD) and UV-visible spectroscopy to establish the spectral properties of its tightly bound flavin adenine dinucleotide (FAD) cofactor. The polypeptide backbone displayed a single circular dichroic minimum at 208 nm and a single maximum at 193 nm. The CD spectrum of bound flavin exhibited a single major negative Cotton peak at 364 nm and two minor negative Cotton peaks at 464 and 495 nm. The protein was reversibly unfolded in 9.8 M urea and refolded in buffer in the presence of excess FAD. The refolded enzyme incorporated FAD and catalyzed full activity. The bound FAD displayed an absorption maximum at 464 nm with an extinction coefficient of epsilon 464 = 11700 M-1 cm-1. Anaerobic reduction with dithionite was complete at 1 equiv. Anaerobic reduction with nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), also was essentially complete at 1 equiv and produced a long-wavelength absorbance band characteristic of an FAD-pyridine nucleotide charge transfer complex. Photochemical bleaching in the presence of ethylenediaminetetraacetic acid (EDTA) followed exponential kinetics. None of the anaerobic reductive titrations produced a spectral intermediate characteristic of a flavin semiquinone, and all reduced enzyme species could be fully reoxidized by oxygen, with full recovery of catalytic activity. Photochemically reduced enzyme was reoxidized by titration with either NADP+ or uridine diphospho N-acetylglucosamine enolpyruvate (UNAGEP). Reoxidation by NADP+ reached a chemical equilibrium, whereas reoxidation by UNAGEP was stoichiometric. Binding of NADP+ or UNAGEP to the oxidized form of the enzyme produced a dead-end complex that could be titrated by following a 10-nm red shift in the absorption spectrum of the bound FAD. The Kd of NADP+ for oxidized enzyme was 0.7 +/- 0.3 microM and the Kd of UNAGEP was 2.7 +/- 0.3 microM. Solvent deuterium isotope effects on binding were observed for both NADP+ and UNAGEP, depending on the pH. At pH 8.5, the HKd/DKd was 2.2 for NADP+ and 3.9 for UNAGEP. No spectral changes were observed in the presence of a 40-fold excess of uridine diphospho N-acetylmuramic acid (UNAM) either aerobically or anaerobically. These studies have identified spectral signals for five steps in the kinetic mechanism, have indicated that product formation is essentially irreversible, and have indicated that hydrogen bonding or protonation contributes significantly to ground-state complex formation with the physiological substrate.

Investigating the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase using lanthanide luminescence spectroscopy.

Lanthanide luminescence was used to examine the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase (GS). These studies revealed the presence of two lanthanide ion binding sites of GS of either adenylylation extrema. Individual emission decay lifetimes were obtained in both H2O and D2O solvent systems, allowing for the determination of the number of water molecules coordinated to each bound Eu3+. The results indicate that there are 4.3 +/- 0.5 and 4.6 +/- 0.5 water molecules coordinated to Eu3+ bound to the n1 site of unadenylylated enzyme, GS0, and fully adenylylated enzyme, GS12, respectively, and that there are 2.6 +/- 0.5 water molecules coordinated to Eu3+ at site n2 for both GS0 and GS12. Energy transfer measurements between the lanthanide donor-acceptor pair Eu3+ and Nd3+, obtained an intermetal distance measurement of 12.1 +/- 1.5 A. Distances between a Tb3+ ion at site n2 and tryptophan residues were also performed with the use of single-tryptophan mutant forms of E. coli GS. The dissociation constant for lanthanide ion binding to site n1 was observed to decrease from Kd = 0.35 +/- 0.09 microM for GS0 to Kd = 0.06 +/- 0.02 microM for GS12. The dissociation constant for lanthanide ion binding to site n2 remained unchanged as a function of adenylylation state; Kd = 3.8 +/- 0.9 microM and Kd = 2.6 +/- 0.7 microM for GS0 and GS12, respectively. Competition experiments indicate that Mn2+ affinity at site n1 decreases as a function of increasing adenylylation state, from Kd = 0.05 +/- 0.02 microM for GS0 to Kd = 0.35 +/- 0.09 microM for GS12. Mn2+ affinity at site n2 remains unchanged (Kd = 5.3 +/- 1.3 microM for GS0 and Kd = 4.0 +/- 1.0 microM for GS12). The observed divalent metal ion affinities, which are affected by the adenylylation state, agrees with other steady-state substrate experiments (Abell LM, Villafranca JJ, 1991, Biochemistry 30:1413-1418), supporting the hypothesis that adenylylation regulates GS by altering substrate and metal ion affinities.

Kinetic and crystallographic studies of Escherichia coli UDP-N-acetylmuramate:L-alanine ligase.

Uridine diphosphate-N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) catalyzes the ATP-dependent ligation of the first amino acid to the sugar moiety of the peptidoglycan precursor. This is an essential step in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Optimal assay conditions for initial velocity studies have been established. Steady-state assays were carried out to determine the effect of various parameters on enzyme activity. Factors studies included: cation specificity, ionic strength, buffer composition and pH. At 37 degrees C and pH 8.0, kcat was equal to 980 +/- 40 min-1, while K(m) values for ATP, UNAM, and L-alanine were, 130 +/- 10, 44 +/- 3, and 48 +/- 6 microM, respectively. Of the metals tested only Mn, Mg, and Co were able to support activity. Sodium chloride, potassium chloride, ammonium chloride, and ammonium sulfate had no effect on activity up to 75 mM levels. The enzyme, in appropriate buffer, was stable enough to be assayed over the pH range of 5.6 to 10.1. pH profiles of Vmax/K(m) for the three substrates and of Vmax were obtained. Crystallization experiments with the enzyme produced two crystal forms. One of these has been characterized by X-ray diffraction as monoclinic, space group C2, with cell dimensions a = 189.6, b = 92.1, c = 75.2 A, beta = 105 degrees, and two 54 kDa molecules per asymmetric unit. It was discovered that the enzyme will hydrolyze ATP in the absence of L-alanine. This L-alanine independent activity is dependent upon the concentrations of both ATP and UNAM; kcat for this activity is less than 4% of the biosynthetic activity measured in the presence of saturating levels of L-alanine. Numerous L-alanine analogs tested were shown to stimulate ATP hydrolysis. A number of these L-alanine analogs produced novel products as accessed by HPLC and mass spectral analysis. All of the L-alanine analogs tested as inhibitors were competitive versus L-alanine.

Design of heterotetrameric coiled coils: evidence for increased stabilization by Glu(-)-Lys(+) ion pair interactions.

Electrostatic interactions between charged amino acids often affect heterospecificity in coiled coils as evidenced by the interaction between the oncoproteins, fos and jun. Such interactions have been successfully exploited in the design of heteromeric coiled coils in a number of laboratories. It has been suggested that heterospecificity in these dimeric coiled-coil systems is driven not by specific electrostatic interactions in the heterodimers but rather by electrostatic repulsion acting to destabilize the homodimer state relative to the heterodimer state. We show that it is possible to design ion pair interactions that directly stabilize the heterotetrameric coiled-coil state. Synthetic peptides were used whose sequences are based on the C-terminal tetramerization domain of Lac repressor, as a model system for four-chain coiled coils (Fairman et al., 1995). These Lac-based peptides, containing either glutamic acid (Lac21E) or lysine (Lac21K) at all b and c heptad positions, only weakly self-associate but, when mixed, afford a highly stable heterotetramer. This study represents the first experimental evidence for the importance of the b and c heptad positions to the stability of coiled coils. Finally, pH dependence and NaCl dependence studies show that heterotetramer stability is driven by ion pair interactions between glutamate and lysine; these interactions contribute about 0.6 kcal/mol of stabilizing free energy for each potential glutamate-lysine pair.

Structural studies of Escherichia coli UDP-N-acetylmuramate:L-alanine ligase.

Uridine diphosphate N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) adds the first amino acid to the sugar moiety of the peptidoglycan precursor, catalyzing one of the essential steps in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Here, we report our studies on the secondary and quaternary structures of UNAM:L-Ala ligase from Escherichia coli. The molecular weight of the purified recombinant enzyme determined by electrospray ionization mass spectrometry agreed well with the molecular weight deduced from the DNA sequence. Through sedimentation equilibrium analysis, we show that the enzyme exists in equilibrium between monomeric and dimeric forms and that the dissociation constant of the dimer, Kd, was determined to be 1.1 +/- 0.4 microM at 37 degrees C and 0.58 +/- 0.30 microM at 4 degrees C. A very similar Kd value was also obtained at 37 degrees C by gel filtration chromatography. The secondary structure of the enzyme was characterized by circular dichroism spectroscopy. No change in the secondary structure was observed between the monomeric and dimeric forms of the enzyme. The activity assays at enzyme concentrations both below and above the determined Kd value lead to the conclusion that the enzyme is active both as dimers and as monomers and that the specific activity is independent of the oligomerization state.

Effect of metal-ligand mutations on phosphoryl transfer reactions catalyzed by Escherichia coli glutamine synthetase.

Glutamine synthetase (GS) converts glutamate to glutamine in the presence of ATP and ammonia and requires two divalent metal ions, designated n1 and n2, for catalysis. The first intermediate, gamma-glutamyl phosphate, is formed during catalysis by the transfer of the gamma-phosphate of ATP to the gamma-carboxylate of glutamate. Efficient phosphoryl transfer between these two negatively charged moieties is thought to be mediated by the n2 metal. To explore the role of the n2 metal in catalysis, histidine 269, a ligand to the n2 metal, was changed to aspartate, asparagine, glutamate, and glutamine by site-directed mutagenesis. All of the mutants bind two manganese ions as determined by EPR titration. The mutations had little effect on the substrate Km's except in the case of H269E which exhibited a Km Glu = 92 mM, a 1000-fold increase compared to that for WT (Km Glu = 70 microM). The ability of these mutants to catalyze phosphoryl transfer to glutamate or to the inhibitor phosphinothricin was examined by rapid quench kinetic experiments. Phosphorylated phosphinothricin was detected by 31P NMR and shown to be produced by both mutants and WT. The rate of phosphoryl transfer to PPT for H269E is reduced 100-fold (0.030 s-1) compared to WT (8 s-1). The extra negative charge around the n2 metal ion contributed by glutamate 269 severely reduces the ability of the n2 metal to mediate efficient glutamate binding in the presence of negatively charged ATP and weakens the interactions between metal ion and the reactants in the transition state, thus resulting in a lower rate of phosphoryl transfer.

Cooperativity and binding in the mechanism of cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) hydrolyzes the sn-2 ester of phospholipids and is believed to be responsible for the receptor-regulated release of arachidonic acid from phospholipid pools. The enzyme was assayed using vesicles containing arachidonate-containing phospholipid substrate, such as 1-palmitoyl-2-arachidonoylphosphatidylcholine (PAPC) or 1-stearoyl-2-arachidonoylphosphatidylinositol (SAPI), dispersed within vesicles of 1,2-dimyristoylphosphatidylmethanol (DMPM). We report here that the enzyme shows an apparent cooperative effect with respect to the mole fraction of arachidonate-containing phospholipids within these covesicles. The data can be fit to a modified Hill equation yielding Hill coefficients, n, of 2-3. This effect is unusual in that it is dependent on the nature of the sn-2 ester as opposed to the phosphoglycerol head group. This cooperativity is independent of both the concentration of glycerol, which greatly increases enzyme activity and stability, and the concentration of calcium, which facilitates the fusion of the covesicles. Surprisingly, 1-palmitoyl-2-arachidonoylphosphatidylethanolamine (PAPE) does not show the same cooperative effect, although the rate at which it is hydrolyzed is much greater when PAPC is present. Moreover, PAPE has a dissociation constant from the active site (KD* = 0.7 mol %) which is comparable to that of PAPC and SAPI (KD* values of 0.3 and 0.3 mol %, respectively). These results are consistent with the presence of an allosteric site that, when occupied, induces a change in the enzyme which facilitates enzymatic hydrolysis. If so, PAPC and SAPI, but not PAPE, must be able to bind to this allosteric site. Alternatively, this effect may result from changes in the physical nature of the bilayer which result upon increasing the bilayer concentration of arachidonate-containing phospholipids. This previously unobserved effect may represent another mechanism by which cells can regulate the activity of cPLA2.

Steady-state kinetic mechanism of Escherichia coli UDP-N-acetylenolpyruvylglucosamine reductase.

The Escherichia coli MurB gene encoding UDP-N-acetylenolpyruvylglucosamine reductase was expressed to a level of approximately 100 mg/L as a fusion construct with maltose binding protein. Rapid affinity purification, proteolysis, and anion exchange chromatography yielded homogeneous enzyme containing 1 mol/mol bound FAD. Enzyme was maximally activated by K+, NH4+, and Rb+ at cation concentrations between 10 and 50 mM. Steady-state enzyme kinetics at pH 8.0 and 37 degrees C revealed weak and strong substrate inhibition by NADPH and UDP-N-acetylenolpyruvylglucosamine, respectively, where the KiS were 910 microM and 73 microM. Substrate inhibition was pH dependent for both substrates. Initial velocity measurements as a function of both substrates produced patterns consistent with a ping pong bi bi double competitive substrate inhibition mechanism. Data at pH 8.0 yielded kinetic constants corresponding to Km,UNAGEP = 24 +/- 3 microM, Ki,UNAGEP = 73 +/- 19 microM, Km,NADPH = 17 +/- 3 microM, Ki,NADPH = 910 +/- 670 microM, and kcat = 62 +/- 3 s-1. A slow anaerobic exchange reaction with thio-NADP+ provided evidence for release of NADP+ in the absence of UNAGEP. Alternate reduced nicotinamide dinucleotides, including NHXDPH, 3'-NADPH, and alpha-NADPH, were substrates, whereas NADH was not. Several nucleotides, including ADP and UDP, were weak inhibitors of the enzyme with inhibition constants between 5 and 97 mM. Various analogs of NADP+, including 3'-NADP+, thio-NADP+, APADP+, NEthDP+, and NHXDP+, were inhibitors of the enzyme with respect to NADPH and yielded inhibition constants in the range of 110-1100 microM. Analogs without the 2'- or 3'-phosphate of NADPH or NADP+ were not substrates or inhibitors. Double inhibition experiments with varied APADP+ and UNAG produced inhibition patterns consistent with mutually exclusive inhibitor binding. The data suggest that NADPH and UNAGEP share a subsite that prevents both molecules from binding at once.

Active recombinant human cytosolic phospholipase A2 is expressed in Escherichia coli.

The cDNA encoding human cytosolic phospholipase A2 (cPLA2) has been subcloned into a prokaryotic pET16b expression vector which also encodes an amino-terminal deca-histidine affinity tag to facilitate purification of the recombinant enzyme. Soluble, active fusion protein, designated His-cPLA2, has been obtained reproducibly from this expression system using the E. coli strain BL21 (DE3). The protein has been purified to homogeneity in four steps and the mass confirmed by electrospray mass spectrometry. His-cPLA2 was characterized by kinetic analysis which demonstrated that the enzyme is similar to native cPLA2 in all respects investigated. Specifically, the enzyme binds to anionic vesicles containing substrate, and acts processively on these vesicles. Enzymatic activity is supported by the presence of Ca2+ and several other divalent metal ions, and is inhibited by several transition metal ions. Finally, the enzyme demonstrates lysophospholipase activity and exhibits a high selectivity for sn-2 arachidonyl esters. This prokaryotic expression system yields moderate amounts of unmodified recombinant His-cPLA2 and is advantageous for rapid production of protein and mutational analyses.

Dimerization of native and C-terminally proteolyzed p56lck tyrosine kinase.

Recombinant p56lck tyrosine kinase was purified to near homogeneity from a baculovirus/insect cell expression system. Treatment with thrombin proteolytically removed the C-terminal 54 amino acids from p56lck. Processed enzyme migrated on sodium dodecyl sulfate (SDS) gels with a M(r) approximately 6,000 lower than intact enzyme. Analytical ultracentrifugation of intact and processed p56lck gave M(r)'s of 62,600 and 56,200, respectively, confirming that the thrombin treated enzyme existed in solution as a processed polypeptide and that there was no anomalous migration in SDS gels due to thrombin treatment. Simultaneous multispeed analysis of sedimentation equilibrium data demonstrated that both intact and processed enzyme can dimerize with a weak binding constant in the range of 200-300 microM. Purified intact p56lck incorporated 2 mol of [32P]P(i) per mole of enzyme. Purified processed p56lck incorporated only 1 mol of [32P]P(i) per mole of enzyme. The loss of 1 mol of [32P]P(i) per mole of enzyme after thrombin deletion of the C-terminus demonstrates that p56lck undergoes autophosphorylation at the C-terminus. The data are consistent with autophosphorylation at tyrosine 505, which has previously been thought to be a regulatory phosphorylation site, but which now must also be considered as an autophosphorylation site.

Probing the catalytic roles of n2-site glutamate residues in Escherichia coli glutamine synthetase by mutagenesis.

The contribution of metal ion ligand type and charge to catalysis and regulation at the lower affinity metal ion site (n2 site) of Escherichia coli glutamine synthetase (GS) was tested by mutagenesis and kinetic analysis. The 2 glutamate residues at the n2 site, E129 and E357, were changed to E129D, E129H, E357H, E357Q, and E357D, representing conservative and nonconservative alterations. Unadenylylated and fully adenylylated enzyme forms were studied. The Mn(2+)-KD values, UV-cis and fluorescence emission properties were similar for all mutants versus WTGS, except E129H. For kinetic determinations with both Mn2+ and Mg2+, nonconservative mutants (E357H, E129H, E357Q) showed lower biosynthetic activities than conservative mutants (E129D, E357D). Relative to WTGS, all the unadenylylated Mn(2+)-activated enzymes showed reduced kcat/Km values for ATP (> 7-fold) and for glutamate (> 10-fold). Of the unadenylylated Mg(2+)-activated enzymes, only E129D showed kinetic parameters competitive with WTGS, and adenylylated E129D was a 20-fold better catalyst than WTGS. We propose the n2-site metal ion activates ADP for departure in the phosphorylation of glutamate by ATP to generate gamma-glutamyl phosphate. Alteration of the charge density at this metal ion alters the transition-state energy for phosphoryl group transfer and may affect ATP binding and/or ADP release. Thus, the steady-state kinetic data suggest that modifying the charge density increases the transition-state energies for chemical steps. Importantly, the data demonstrate that each ligand position has a specialized spatial environment and the charge of the ligand modulates the catalytic steps occurring at the metal ion. The data are discussed in the context of the known X-ray structures of GS.

Complete assignment of disulfide bonds in bovine dopamine beta-hydroxylase.

Peptide mapping, chemical sequencing, microbore HPLC/electrospray ionization mass spectrometry (LC/ESI/MS), and matrix-assisted laser desorption mass spectrometry (MALDI/MS) were used to identify the sites of intra- and intermolecular disulfide linkages in bovine dopamine beta-hydroxylase. The enzyme contains 14 cysteines and seven disulfides per monomer. Edman sequencing of tryptic and peptic peptides determined linkages at positions Cys140-Cys582, Cys218-Cys269, Cys255-Cys281, Cys452-Cys474, Cys514-Cys514, and Cys516-Cys516, where cysteines at positions 514 and 516 on one monomer disulfide pair with their homologs on a second monomer. These linkages were confirmed by LC/ESI/MS and MALDI/MS. Further analysis by LC/ESI/MS and MALDI/MS identified linkages at positions Cys376-Cys489 and Cys380-Cys551. Cysteines 140 and 582 form a disulfide linkage that folds the C-terminus back in proximity to the N-terminus. The remaining intramolecular disulfides occur along two separate internal regions of the protein. The density of histidine residues in these two regions suggests binding sites for two Cu2+ atoms per monomer. In addition, previously identified amino acids that react with mechanism-based inactivators occur in these two regions. We propose that these five internal disulfide bonds define two Cu2+ binding domains that make up the active site of a dopamine beta-hydroxylase monomer. Considering previous data on the location of glycosylation sites, mechanism-based inactivation sites, and the disulfide linkages presented here, the data suggest an overall topology were the N- and C-termini are in close proximity and are solvent exposed and where the Cu2+ binding sites are buried in two interior domains stabilized by five disulfide bonds.

Bifunctional peptidylglcine alpha-amidating enzyme requires two copper atoms for maximum activity.

The conversion of C-terminal glycine-extended peptides to C-terminal alpha-amidated peptides occurs in two distinct reactions, both of which are catalyzed by bifunctional peptidylglycine alpha-amidating enzyme. The first step is the alpha-hydroxylation of the C-terminal glycine residue and the second step is the dealkylation of the alpha-hydroxyglycine-extended peptide to the alpha-amidated peptide and glyoxylate. We show that the bifunctional enzyme requires 1.9 +/- 0.2 mol of copper/mol of enzyme for maximal dansyl-Tyr-Lys-Gly amidation activity under the conditions of high enzyme concentration (approximately 80 microM) required to measure initial rates for this poor substrate. The enzyme, as purified, contains a substoichiometric amount of copper and has only trace levels of amidation activity. Addition of exogenous Cu(II) ions stimulates amidation activity approximately 3000-fold at the optimum copper stoichiometry and the enzyme is then inhibited by excess Cu(II). No stimulation of amidation activity is observed upon the addition of the following divalent metal ions: Mn(II), Fe(II), Ni(II), Cd(II), and the oxovanadium cation, VO(II). The enzyme-catalyzed dealkylation of alpha-hydroxyhippuric acid to benzamide shows no dependence on copper, indicating that the copper dependence of the amidation reaction must be attributed to a copper dependence in peptide alpha-hydroxylation.

Regeneration of catalytic activity of glutamine synthetase mutants by chemical activation: exploration of the role of arginines 339 and 359 in activity.

In order to understand the nature of ATP and L-glutamate binding to glutamine synthetase, and the involvement of Arg 339 and Arg 359 in catalysis, these amino acids were changed to cysteine via site-directed mutagenesis. Individual mutations (Arg-->Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the Km values for the substrates ATP and glutamate were elevated substantially above the values for wild-type (WT) enzyme. Each cysteine was in turn chemically modified to an arginine "analog" to attempt to "rescue" catalytic activity by covalent modification; 2-chloroacetamidine (CA) (producing a thioether) and 2,2'-dithiobis (acetamidine)(DTBA) (producing a disulfide) were the reagents used to effect these chemical transformations. Upon reaction with CA, both R339C and R359C mutants showed a significant regain of catalytic activity (50% and 70% of WT, respectively) and a drop in Km value for ATP close to that for WT enzyme. With DTBA, chemically modified R339C had a greater kcat than WT glutamine synthetase, but chemically modified R359C only regained a small amount of activity. Modification with DTBA was quantitative for each mutant and each modified enzyme had similar Km values for both ATP and glutamate. The high catalytic activity of DTBA-modified R339C could be reversed to that of unmodified R339C by treatment with dithiothreitol, as expected for a modified enzyme containing a disulfide bond. Modification of each cysteine-containing mutant to a lysine "analog" was accomplished using 3-bromopropylamine (BPA).(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic and mutagenic studies of the role of the active site residues Asp-50 and Glu-327 of Escherichia coli glutamine synthetase.

The role of Asp-50 and Glu-327 of Escherichia coli glutamine synthetase in catalysis and substrate binding has been interrogated by construction of site-directed mutants at these positions. Steady-state and rapid-quench kinetic methods were used to elucidate contributions of Asp-50 and Glu-327 to the Km values of all three substrates, ATP, glutamate, and NH4+, as well as to the enzymatic kcat value. Kinetic constants were obtained for the D50A enzyme using both Mg2+ and Mn2+ as activating metal ions; the data reveal that Asp-50 has a significant role in both substrate binding and catalysis as reflected by the increases in the Km values for NH4+ and the destabilization of both the ground state and the transition state for phosphoryl transfer. The D50E mutant was found to have activity with Mn2+ but very low activity with Mg2+, the physiologically important metal ion. The kcat/Km values for all three substrates were substantially altered by changing Asp to Glu. The steady-state results for the E327A mutant indicate a decreased kcat/Km value for NH4+ compared to that of the wild-type enzyme. The E327A-Mg2+ enzyme destabilizes the ground state of the ternary complex (E-ATP-Glu-NH4+) and the transition state for phosphoryl transfer while the E327A-Mn2(+)-enzyme provides greater stabilization for the ATP and glutamate complexes but destabilizes phosphoryl transfer steps in the ternary complex. Overall, these results suggest that Asp-50 is likely involved in binding NH4+ and may also play a role in catalyzing deprotonation of NH4+ to form NH3.(ABSTRACT TRUNCATED AT 250 WORDS)

A model for oxidative modification of glutamine synthetase, based on crystal structures of mutant H269N and the oxidized enzyme.

Proteolytic degradation of glutamine synthetase (GS) in Escherichia coli is known to follow "marking" by oxidative modification. At an early stage of the degradative pathway, oxidation of His 269 and Arg 344 abolishes GS enzymatic activity. We propose a mechanism for the early stage of oxidative inactivation of GS on the basis of the crystal structure of H269N and tryptophan fluorescence spectra of H269N and H269NR344Q: (1) Oxidation of Arg 344, adjacent to the n2 metal ion site, decreases ATP binding. (2) Oxidation of His 269 to Asn destroys the n2 site, consistent with the function of His 269 as a ligand for the n2 metal. (3) Loss of Mn2+ at the n2 site destroys the integrity of the ATP binding site. (4) Destruction of the ATP site results in the observed low enzymatic activity of H269N and H269NR344Q. During later stages of oxidative modification, the n1 metal ion site is destroyed and the active site of the enzyme becomes flexible as suggested by X-ray data collected from an oxidized crystal of GS. Thus, studies of mutant and oxidized enzymes confirm that there are at least two stages of oxidative modification of GS. These studies suggest that the early modification occurs at the n2 metal ion site, eliminating enzyme activity, and the later modification occurs at the n1 metal ion site, relaxing the GS structure, perhaps enabling proteolytic degradation. These studies also illuminate the differing roles of the two bound metal ions: the tightly bound n1 ion enhances the stability of the catalytically active conformation, and the less tightly bound n2 ion participates in ATP binding.