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.

Characterization of NADP+ binding to perdeuterated MurB: backbone atom NMR assignments and chemical-shift changes.

Backbone-atom resonances have been assigned for both the substrate-free and the NADP+-complexed forms of UDP-N-acetylenolpyruvylglucosamine reductase (MurB), a monomeric, 347-residue (38.5 kDa) flavoenzyme essential for bacterial cell-wall biosynthesis. NMR studies were performed using perdeuterated, uniformly 13C/15N-labeled samples of MurB. In the case of substrate-free MurB, one or more backbone atoms have been assigned for 334 residues (96%). The assigned backbone atoms include 309 1HN and 15N atoms (94%), 315 13CO atoms (91%), 331 13C(alpha) atoms (95%), and 297 13C(beta) atoms (93%). For NADP+-complexed MurB, one or more backbone atoms have been assigned for 313 residues (90%); these include 283 1HN and 15N atoms (86%), 305 13CO atoms (88%), 310 13C(alpha) atoms (89%), and 269 13C(beta) atoms (84%). The strategies used for obtaining resonance assignments are described in detail. Information on the secondary structure in solution for both the substrate-free and NADP+-complexed forms of the enzyme has been derived both from 13C(alpha) and 13C(beta) chemical-shift deviations from random-coil values and from 1HN-1HN NOEs. These data are compared to X-ray crystallographic structures of substrate-free MurB and MurB complexed with the UDP-N-acetylglucosamine enolpyruvate (UNAGEP) substrate. NADP+ binding induces significant chemical-shift changes in residues both within the known UNAGEP and FAD binding pockets and within regions known to undergo conformational changes upon UNAGEP binding. The NMR data indicate that NADP+ and UNAGEP utilize the same binding pocket and, furthermore, that the binding of NADP+ induces structural changes in MurB. Finally, many of the residues within the UNAGEP/NADP+ binding pocket were difficult to assign due to dynamic processes which weaken and/or broaden the respective resonances. Overall, our results are consistent with MurB having a flexible active site.

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.

Crystallization and preliminary crystallographic analysis of E. coli uridine 5'-diphospho-N-acetylenolpyruvylglucosamine reductase in two new crystal forms.

Uridine 5'-diphospho-N-acetylenolpyruvylglucosamine reductase (MurB), the second enzyme in the peptidoglycan synthetic pathway of Escherichia coli, has been crystallized in two previously unreported forms, one orthorhombic and the other monoclinic. MurB (molecular mass 38 kDa) crystallizes in a range of conditions that utilize polyethylene glycol fractions as precipitants, and crystals can be grown with or without the enzyme's substrate, uridine 5'-diphospho-N-acetylenolpyruvylglucosamine. X-ray diffraction from crystals of the orthorhombic form extends to 2 A resolution and shows the symmetry and systematic absences of space group P2(1)2(1)2(1). These crystals show significant variations in cell dimensions at room temperature and at 100 K. A crystal used to collect a 2.0 A resolution data set at a synchrotron source showed cell dimensions at ca 100 K of a = 51.0, b = 79.3 and c = 87.1 A, indicating one molecule peroasymmetric unit. The monoclinic crystals scatter X-rays to 3.0 A resolution consistent with space group P2(1), unit-cell dimensions (ca 100 K) a = 50.7, b = 92.4, c = 85.5 A, and beta = 104 degrees, and two molecules per asymmetric unit. Mercury derivatives have been prepared with both orthorhombic and monoclinic forms, and efforts are underway to exploit these derivatives to determine the structure of this protein.

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.

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.

Supramolecular self-assembly of Escherichia coli glutamine synthetase: characterization of dodecamer stacking and high order association.

Dodecameric glutamine synthetase (GS) from bacteria is formed from two face-to-face hexameric rings of identical subunits. These highly symmetrical aggregates from some bacteria, including Escherichia coli, "stack" in the presence of Zn2+ and other divalent ions to generate protein tubes (phase I) and subsequently associate side-to-side to yield "cables" and nonspecific aggregates (phase II). In order to understand the molecular mechanisms of recognition leading to this macromolecular self-assembly, the effects of solution conditions on the kinetics of these processes have been studied. These reactions have been monitored by changes in light scattering and by electron microscopy. Conditions have been established for isolation of phases I and II. At 0.04 mg of GS/mL, pH 7.0, 100 mM KCl, and 1 mM Mn2+, 25 degrees C, minimal side-to-side aggregation occurs, and the stacking reaction follows second-order kinetics, with respect to GS, at low extent of reaction. The second-order rate constants determined for phase I, initiated by Zn2+ or Co2+, demonstrate a pH optimum at 7.0-7.25, whereas phase II is favored at pHs below 6.5. The pH profile for the stacking reaction suggests that His residues are involved, and modification of 2-3 histidines/subunit with diethyl pyrocarbonate (DEPC) is sufficient to completely inhibit metal-dependent dodecamer stacking. The effect of ionic strength on GS stacking was also studied. Although hydrophobic interactions have previously been assumed to dominate this protein-protein association, both phase I and phase II of the assembly are inhibited by KCl and NaCl, suggesting that ionic interactions also play an essential role.(ABSTRACT TRUNCATED AT 250 WORDS)

Supramolecular self-assembly of glutamine synthetase: mutagenesis of a novel intermolecular metal binding site required for dodecamer stacking.

Dodecameric glutamine synthetase (GS) from Escherichia coli assembles into highly ordered supramolecular protein tubes in the presence of several divalent metal ions. The molecular mechanism for this metal-induced self-assembly of the E. coli GS has been studied by molecular modeling and site-directed mutagenesis. The X-ray crystal structure of the nearly identical Salmonella typhimurium GS has been used to construct a model of the "stacked" complex between two dodecamers. A complementary fit, based on steric constraints, reveals a possible interaction between the N-terminal helices from adjacent dodecamers. The amino acid side chains of His and Met residues within the helices from each of the subunits of one face of a dodecamer lie within approximately 3.5 A of the analogous side chains in the subunits from the adjacent dodecamer in the stacked complex. His-4, Met-8, and His-12 from adjacent helices provide potential ligands for a binuclear metal binding site. Replacement of each of these surface residues with aliphatic amino acids has negligible effects on the enzymatic activity, the regulation of activity via adenylylation, and gross dodecameric structure. However, the rate and extent of metal ion-mediated self-assembly of GS tubules are reduced to < 2% of the wild-type protein in the single mutants H4A, H12L, and H12D. The M8L mutant demonstrates a 3-fold decrease in the bimolecular rate constant for stacking, but electron microscopy indicates that this mutant does form stacked tubes. The cysteine-containing mutants H4C, M8C, and H12C were also constructed.(ABSTRACT TRUNCATED AT 250 WORDS)