Mechanistic inferences from the binding of ligands to LpxC, a metal-dependent deacetylase

The metal-dependent deacetylase LpxC catalyzes the first committed step of lipid A biosynthesis in Gram-negative bacteria. Accordingly, LpxC is an attractive target for the development of inhibitors that may serve as potential new antibiotics for the treatment of Gram-negative bacterial infections. Here, we report the 2.7 A resolution X-ray crystal structure of LpxC complexed with the substrate analogue inhibitor TU-514 and the 2.0 A resolution structure of LpxC complexed with imidazole. The X-ray crystal structure of LpxC complexed with TU-514 allows for a detailed examination of the coordination geometry of the catalytic zinc ion and other enzyme-inhibitor interactions in the active site. The hydroxamate group of TU-514 forms a bidentate chelate complex with the zinc ion and makes hydrogen bond interactions with conserved active site residues E78, H265, and T191. The inhibitor C-4 hydroxyl group makes direct hydrogen bond interactions with E197 and H58. Finally, the C-3 myristate moiety of the inhibitor binds in the hydrophobic tunnel of the active site. These intermolecular interactions provide a foundation for understanding structural aspects of enzyme-substrate and enzyme-inhibitor affinity. Comparison of the TU-514 complex with cacodylate and imidazole complexes suggests a possible substrate diphosphate binding site and highlights residues that may stabilize the tetrahedral intermediate and its flanking transition states in catalysis. Evidence of a catalytic zinc ion in the native zinc enzyme coordinated by H79, H238, D242, and two water molecules with square pyramidal geometry is also presented. These results suggest that the native state of this metallohydrolase may contain a pentacoordinate zinc ion, which contrasts with the native states of archetypical zinc hydrolases such as thermolysin and carboxypeptidase A.     

Inhibition of human arginase I by substrate and product analogues

Human arginase I is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of l-arginine to generate l-ornithine and urea. We demonstrate that N-hydroxy-l-arginine (NOHA) binds to this enzyme with K_d = 3.6 μM, and nor-N-hydroxy-l-arginine (nor-NOHA) binds with K_d = 517 nM (surface plasmon resonance) or K_d ≈ 50 nM (isothermal titration calorimetry). Crystals of human arginase I complexed with NOHA and nor-NOHA afford 2.04 and 1.55 Å resolution structures, respectively, which are significantly improved in comparison with previously-determined structures of the corresponding complexes with rat arginase I. Higher resolution structures clarify the binding interactions of the inhibitors. Finally, the crystal structure of the complex with l-lysine (K_d = 13 μM) is reported at 1.90 Å resolution. This structure confirms the importance of hydrogen bond interactions with inhibitor α-carboxylate and α-amino groups as key specificity determinants of amino acid recognition in the arginase active site.     

Thermodynamics and stoicheiometry of the binding of substrate analogues to uricase.

The subunit composition, metal content, substrate-analogue binding and thermal stability of Aspergillus flavus uricase were determined. A. flavus uricase is a tetramer and contains no copper, iron or any other common prosthetic group. Analytical-gel-filtration and equilibrium-dialysis experiments showed one binding site per subunit for urate analogues. The free energy of xanthine binding was -30.5 kJ (-7.3 kcal)/mol of subunit by equilibrium dialysis and -30.1 kJ (-7.2 kcal)/mol of subunit by microcalorimetry. The enthalpy change for xanthine binding was -15.9 kJ (-3.8 kcal)/mol of subunit when determined from the temperature-dependence of the equilibrium constant and -18.0 kJ (-4.3 kcal)/mol of subunit when measured microcalorimetrically. The thermal inactivation rate of A. flavus uricase increases as protein concentration is decreased. This concentration-dependent instability is not due to subunit dissociation.     

The pH-dependence of the binding of dihydrofolate and substrate analogues to dihydrofolate reductase from Escherichia coli

The interaction of dihydrofolate reductase (EC 1.5.1.3) from Escherichia coli with dihydrofolate and folate analogues has been studied by means of binding and spectroscopic experiments. The aim of the investigation was to determine the number and identity of the binary complexes that can form, as well as pKa values for groups on the ligand and enzyme that are involved with complex formation. The results obtained by ultraviolet difference spectroscopy indicate that, when bound to the enzyme, methotrexate and 2,4-diamino-6,7-dimethylpteridine exist in their protonated forms and exhibit pKa values for their N-1 nitrogens of above 10.0. These values are about five pH units higher than those for the compounds in free solution. The binding data suggest that both folate analogues interact with the enzyme to yield a protonated complex which may be formed by reaction of ionized enzyme with protonated ligand and/or protonated enzyme with unprotonated ligand. The protonated complex formed with 2,4-diamino-6,7-dimethylpteridine can undergo further protonation to form a protonated enzyme-protonated ligand complex, while that formed with methotrexate can ionize to give an unprotonated complex. A group on the enzyme with a pKa value of about 6.3 is involved with the interactions. However, the ionization state of this group has little effect on the binding of dihydrofolate to the enzyme. For the formation of an enzyme-dihydrofolate complex it is essential that the N-3/C-4 amide of the pteridine ring of the substrate be in its neutral form. It appears that dihydrofolate is not protonated in the binary complex.     

Modelling of substrate binding to 3-phosphoglycerate kinase with analogues of 3-phosphoglycerate

Two short analogues of 3-phosphoglycerate, -OOC-CHOH-CH2-O-PO32-, phosphonolactate, (-OOC-CHOH-CH2-PO32-) and arsonolactate (-OOC-CHOH-CH2-AsO32-) have been tested with 3-phosphoglycerate kinase. None of these served as substrate for the kinase reaction, unlike the previously studied [Orr, G. A. & Knowles, J. R. (1974) Biochem. J. 141, 721-723] analogues -OOC-CHOH-CH2-CH2-PO32- and -OOC-CHOH-CH2-CH2-AsO32-, which are isosteric with 3-phosphoglycerate. Thus, a decrease in the substrate size and the accompanying stereochemical changes cannot be tolerated by the catalytic mechanism. Instead, both analogues acted as relatively poor competitive inhibitors with respect to both 3-phosphoglycerate and MgATP. AT pH 8.5 and 20 degrees C, the inhibitory constants (Ki) of phosphonolactate and arsnolactate against both substrates are 17 +/- 5 mM and 30 +/- 7 mM, respectively. Surprisingly, however, both analogues proved to be more effective than either 3-phosphoglycerate or its isosteric analogues in protecting the enzyme against modification of its fast-reacting thiols. This comparison suggests that the shorter analogues bind differently, and that the catalytic mechanism demands a precise fitting of the -CH2-O-PO32- segment of the substrate.     

Mechanistic inferences from the crystal structure of fumarylacetoacetate hydrolase with a bound phosphorus-based inhibitor

Fumarylacetoacetate hydrolase (FAH) catalyzes the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate to yield fumarate and acetoacetate as the final step of Phe and Tyr degradation. This unusual reaction is an essential human metabolic function, with loss of FAH activity causing the fatal metabolic disease hereditary tyrosinemia type I (HT1). An enzymatic mechanism involving a catalytic metal ion, a Glu/His catalytic dyad, and a charged oxyanion hole was previously proposed based on recently determined FAH crystal structures. Here we report the development and characterization of an FAH inhibitor, 4-(hydroxymethylphosphinoyl)-3-oxo-butanoic acid (HMPOBA), that competes with the physiological substrate with a K(i) of 85 microM. The crystal structure of FAH complexed with HMPOBA refined at 1.3-A resolution reveals the molecular basis for the competitive inhibition, supports the proposed formation of a tetrahedral alkoxy transition state intermediate during the FAH catalyzed reaction, and reveals a Mg(2+) bound in the enzyme's active site. The analysis of FAH structures corresponding to different catalytic states reveals significant active site side-chain motions that may also be related to catalytic function. Thus, these results advance the understanding of an essential catabolic reaction associated with a fatal metabolic disease and provide insight into the structure-based development of FAH inhibitors.     

Mechanism of binding of substrate analogues to tryptophan indole-lyase: studies using rapid-scanning and single-wavelength stopped-flow spectrophotometry

We have examined the binding of oxindolyl-L-alanine, (3R)-2,3-dihydro-L-tryptophan, L-homophenylalanine, and N1-methyl-L-tryptophan to tryptophan indole-lyase (tryptophanase) from Escherichia coli by using rapid-scanning and single-wavelength stopped-flow kinetic techniques. Rate constants for the reactions were determined by fitting the concentration dependencies of relaxations to either linear (pseudo-first-order) or hyperbolic (rapid second-order followed by slow first-order) equations. The reaction with oxindolyl-L-alanine forms a quinonoid intermediate that exhibits a strong peak at 506 nm. This species is formed more rapidly than with the other analogues (84.5 s-1) and is reprotonated very slowly (0.2 s-1). Reaction with L-homophenylalanine also forms a quinonoid intermediate with a strong peak at 508 nm, but the rate constant for its formation is slower (6.9 s-1). The reaction with L-homophenylalanine exhibits a transient intermediate absorbing at about 340 nm that decays at the same rate as the quinonoid peak forms and that may be a gem-diamine. Tryptophan indole-lyase reacts with (3R)-2,3-dihydro-L-tryptophan much more slowly to form a moderately intense quinonoid peak at 510 nm, and a transient intermediate absorbing at about 350 nm is also formed. The species formed in the reaction of N1-methyl-L-tryptophan exhibits a peak at 425 nm and a very weak quinonoid absorption peak at 506 nm, which is formed at less than 4 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of substrate and substrate analogues on the trinitrophenylation of myosin

The effects of ATP, ATP analogues, Mg²⁺ and actin on the trinitrophenylation of myosin and on the enzymic properties of trinitrophenylated samples were studied.

1. 1. Trinitrophenylation of myosin was inhibited by the presence of ATP and its analogues during the treatment in the order ADP > ATP > pyrophosphate > AMP.

2. 2. The alteration of the enzymic properties due to trinitrophenylation of myosin was prevented by the presence of ATP or ADP and somewhat less by that of pyrophosphate and AMP during trinitrophenylation, but only if Mg²⁺ was also present.

3. 3. Neither the degree of trinitrophenylation nor the enzymic properties of the trinitrophenylated myosin were influenced by the presence of actin during the treatment of myosin.

Mechanistic investigation of medium-chain fatty acyl-CoA dehydrogenase utilizing 3-indolepropionyl/acryloyl-CoA as chromophoric substrate analogues.

The CoA derivative 3-indolepropionyl-CoA (IPCoA) serves as a competent pseudosubstrate for the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction. The reaction product trans-3-indoleacryloyl-CoA (IACoA) exhibits a characteristic UV-vis absorption spectrum with lambda max = 367 nm and epsilon 367 = 26,500 M-1 cm-1. The chromophoric nature of IACoA allows us to measure the direct conversion of substrate to product (at 367 nm) without recourse to absorption signals for either the enzyme-bound flavin or the coupling electron acceptors, as well as probe the enzyme site environment. The interaction of IACoA with medium chain fatty acyl-CoA dehydrogenase (MCAD)-FAD is characterized by resultant (spectra of the mixture minus the individual components) absorption peaks at 490, 417, and 355 nm. These absorption peaks increase in magnitude as the pH of the buffer media decreases. Transient kinetic analysis for the interaction of MCAD-FAD with IACoA suggests that the formation of the enzyme-IACoA complex proceeds in two steps. The first (fast) step involves the formation of an E-IACoA collision complex, which [formula: see text] is isomerized (concomitant with changes in the protein structure) to an E*-IACoA complex in the second (slow) step. We have studied the effect of pH on Kc, k2, and k-2. While Kc shows practically no dependence on pH (within a 2-fold variation between pH 6.0 and 9.5), k2 and k-2 show a strong dependence on pH. Both k2 and k-2 exhibit a sigmoidal dependence on the pH of the buffer media, with pKa's of 7.53 and 8.30, respectively. In accordance with the model presented herein, the pKa of 7.53 represents an enzyme site group which is involved in the interaction with IACoA within the E-IACoA collision complex. This pKa is perturbed to 8.30 upon isomerization of the collision complex. The pH-dependent changes in k2 and k-2 are such that the equilibrium distribution between E-IACoA and E*-IACoA is favored to the latter complex (by about 20-fold) at lower pH than at higher pH. A cumulative account of the spectral, kinetic, and thermodynamic properties of the enzyme-IACoA complexes has allowed us delineate the microscopic pathway by which the E-IACoA isomerization (presumably via protein conformational changes) is coupled to the proton equilibration steps.     

Determination of the substrate binding mode to the active site iron of (S)-2-hydroxypropylphosphonic acid epoxidase using 17O-enriched substrates and substrate analogues

(S)-2-Hydroxypropylphosphonic acid epoxidase (HppE) is an O2-dependent, nonheme Fe(II)-containing oxidase that converts (S)-2-hydroxypropylphosphonic acid ((S)-HPP) to the regio- and enantiomerically specific epoxide, fosfomycin. Use of (R)-2-hydroxypropylphosphonic acid ((R)-HPP) yields the 2-keto-adduct rather than the epoxide. Here we report the chemical synthesis of a range of HPP analogues designed to probe the basis for this specificity. In past studies, NO has been used as an O2 surrogate to provide an EPR probe of the Fe(II) environment. These studies suggest that O2 binds to the iron, and substrates bind in a single orientation that strongly perturbs the iron environment. Recently, the X-ray crystal structure showed direct binding of the substrate to the iron, but both monodentate (via the phosphonate) and chelated (via the hydroxyl and phosphonate) orientations were observed. In the current study, hyperfine broadening of the homogeneous S = 3/2 EPR spectrum of the HppE-NO-HPP complex was observed when either the hydroxyl or the phosphonate group of HPP was enriched with 17O (I = 5/2). These results indicate that both functional groups of HPP bind to Fe(II) ion at the same time as NO, suggesting that the chelated substrate binding mode dominates in solution. (R)- and (S)-analogue compounds that maintained the core structure of HPP but added bulky terminal groups were turned over to give products analogous to those from (R)- and (S)-HPP, respectively. In contrast, substrate analogues lacking either the phosphonate or hydroxyl group were not turned over. Elongation of the carbon chain between the hydroxyl and phosphonate allowed binding to the iron in a variety of orientations to give keto and diol products at positions determined by the hydroxyl substituent, but no stable epoxide was formed. These studies show the importance of the Fe(II)-substrate chelate structure to active antibiotic formation. This fixed orientation may align the substrate next to the iron-bound activated oxygen species thought to mediate hydrogen atom abstraction from the nearest substrate carbon.     

Probing binding requirements of NAD kinase with modified substrate (NAD) analogues

Synthesis of novel NAD(+) analogues that cannot be phosphorylated by NAD kinase is reported. In these analogues the C2' hydroxyl group of the adenosine moiety was replaced by fluorine in the ribo or arabino configuration (1 and 2, respectively) or was inverted into arabino configuration to give compound 3. Compounds 1 and 2 showed inhibition of human NAD kinase, whereas analogue 3 inhibited both the human and Mycobacterium tuberculosis NAD kinase. An uncharged benzamide adenine dinucleotide (BAD) was found to be the most potent competitive inhibitor (K(i)=90 microM) of the human enzyme reported so far.     

Structural and kinetic analysis of substrate binding to the sialyltransferase Cst-II from Campylobacter jejuni

Sialic acids play important roles in various biological processes and typically terminate the oligosaccharide chains on the cell surfaces of a wide range of organisms, including mammals and bacteria. Their attachment is catalyzed by a set of sialyltransferases with defined specificities both for their acceptor sugars and the position of attachment. However, little is known of how this specificity is encoded. The structure of the bifunctional sialyltransferase Cst-II of the human pathogen Campylobacter jejuni in complex with CMP and the terminal trisaccharide of its natural acceptor (Neu5Ac-α-2,3-Gal-β-1,3-GalNAc) has been solved at 1.95 Å resolution, and its kinetic mechanism was shown to be iso-ordered Bi Bi, consistent with its dual acceptor substrate specificity. The trisaccharide acceptor is seen to bind to the active site of Cst-II through interactions primarily mediated by Asn-51, Tyr-81, and Arg-129. Kinetic and structural analyses of mutants modified at these positions indicate that these residues are critical for acceptor binding and catalysis, thereby providing significant new insight into the kinetic and catalytic mechanism, and acceptor specificity of this pathogen-encoded bifunctional GT-42 sialyltransferase.     

Crystal structure of lactaldehyde dehydrogenase from Escherichia coli and inferences regarding substrate and cofactor specificity

Aldehyde dehydrogenases catalyze the oxidation of aldehyde substrates to the corresponding carboxylic acids. Lactaldehyde dehydrogenase from Escherichia coli (aldA gene product, P25553) is an NAD(+)-dependent enzyme implicated in the metabolism of l-fucose and l-rhamnose. During the heterologous expression and purification of taxadiene synthase from the Pacific yew, lactaldehyde dehydrogenase from E. coli was identified as a minor (相似文献   

Thermodynamics of substrate binding to the chaperone SecB

The thermodynamics of binding of unfolded polypeptides to the chaperone SecB was investigated in vitro by isothermal titration calorimetry and fluorescence spectroscopy. The substrates were reduced and carboxamidomethylated forms of RNase A, BPTI, and alpha-lactalbumin. SecB binds both fully unfolded RNase A and BPTI as well as compact, partially folded disulfide intermediates of alpha-lactalbumin, which have 40-60% of native secondary structure. The heat capacity changes observed on binding the reduced and carboxamidomethylated forms of alpha-lactalbumin, BPTI, and RNase A were found to be -0.10, -0.29, and -0.41 kcal mol(-1) K(-1), respectively, and suggest that between 7 and 29 residues are buried upon substrate binding to SecB. In all cases, binding occurs with a stoichiometry of one polypeptide chain per monomer of SecB. There is no evidence for two separate types of binding sites for positively charged and hydrophobic ligands. Spectroscopic and proteolysis protection studies of the binding of SecB to poly-L-Lys show that binding of highly positively charged peptide ligands to negatively charged SecB leads to charge neutralization and subsequent aggregation of SecB. The data are consistent with a model where SecB binds substrate molecules at an exposed hydrophobic cleft. SecB aggregation in the absence of substrate is prevented by electrostatic repulsion between negatively charged SecB tetramers.