Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 1. Crystallography and site-directed mutagenesis of metal binding sites.

The structure and function of the xylose (glucose) isomerase from Actinoplanes missouriensis have been analyzed by X-ray crystallography and site-directed mutagenesis after cloning and overexpression in Escherichia coli. The crystal structure of wild-type enzyme has been refined to an R factor of 15.2% against diffraction data to 2.2-A resolution. The structures of a number of binary and ternary complexes involving wild-type and mutant enzymes, the divalent cations Mg2+, Co2+, or Mn2+, and either the substrate xylose or substrate analogs have also been determined and refined to comparable R factors. Two metal sites are identified. Metal site 1 is four-coordinated and tetrahedral in the absence of substrate and is six-coordinated and octahedral in its presence; the O2 and O4 atoms of linear inhibitors and substrate bind to metal 1. Metal site 2 is octahedral in all cases; its position changes by 0.7 A when it binds O1 of the substrate and by more than 1 A when it also binds O2; these bonds replace bonds to carboxylate ligands from the protein. Side chains involved in metal binding have been substituted by site-directed mutagenesis. The biochemical properties of the mutant enzymes are presented. Together with structural data, they demonstrate that the two metal ions play an essential part in binding substrates, in stabilizing their open form, and in catalyzing hydride transfer between the C1 and C2 positions.     

Inhibition of chymotrypsin by peptidyl trifluoromethyl ketones: determinants of slow-binding kinetics

A series of seven peptidyl trifluoromethyl ketone (TFK) inhibitors of chymotrypsin have been prepared which differ at the P1 and P2 subsites. Inhibition equilibria and kinetics of association and dissociation with chymotrypsin have been measured. The association rate of Ac-Phe-CF3 was measured at enzyme concentrations between 8 nM and 117 microM in order to examine the relation between the ketone/hydrate equilibrium of trifluoromethyl ketones and the "slow binding" by these inhibitors. The association rate decreases at high enzyme concentrations, indicating that TFK ketone is the reactive species and that conversion of TFK hydrate to ketone becomes rate limiting under these conditions. Inhibitors with hydrophobic side chains at P2 bind more tightly but more slowly to chymotrypsin, indicating that formation of van der Waals contacts between the P2 side chain and the His 57 and Ile 99 side chains of chymotrypsin is a relatively slow process. Inhibitor properties were compared to the Michaelis-Menten kinetic constants of a homologous series of peptide methyl ester and peptide amide substrates. Plots of log Ki vs log (kcat/Km) are linear with slopes of 0.65 +/- 0.2, indicating that these inhibitors are able to utilize 65% of the total binding energy between chymotrypsin and its hydrolytic transition state.     

Kinetic and crystallographic studies of glucopyranose spirohydantoin and glucopyranosylamine analogs inhibitors of glycogen phosphorylase

Glycogen phosphorylase (GP) is currently exploited as a target for inhibition of hepatic glycogenolysis under high glucose conditions. Spirohydantoin of glucopyranose and N-acetyl-beta-D-glucopyranosylamine have been identified as the most potent inhibitors of GP that bind at the catalytic site. Four spirohydantoin and three beta-D-glucopyranosylamine analogs have been designed, synthesized and tested for inhibition of GP in kinetic experiments. Depending on the functional group introduced, the K(i) values varied from 16.5 microM to 1200 microM. In order to rationalize the kinetic results, we determined the crystal structures of the analogs in complex with GP. All the inhibitors bound at the catalytic site of the enzyme, by making direct and water-mediated hydrogen bonds with the protein and by inducing minor movements of the side chains of Asp283 and Asn284, of the 280s loop that blocks access of the substrate glycogen to the catalytic site, and changes in the water structure in the vicinity of the site. The differences observed in the Ki values of the analogs can be interpreted in terms of variations in hydrogen bonding and van der Waals interactions, desolvation effects, ligand conformational entropy, and displacement of water molecules on ligand binding to the catalytic site.     

Mobility of the spin-labeled side chains of some novel antifolate inhibitors in their complexes with dihydrofolate reductase

Four spin-labeled inhibitors of dihydrofolate reductase (DHFR) have been synthesized, each of which has the 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) reporting group at a different distance from the 2,4-diaminopyrimidine moiety by which the inhibitors are anchored and oriented in the active site. Inhibitors in which the TEMPO group is attached by a short side chain are weakly bound to DHFR from bacteria (Streptococcus faecium and Lactobacillus casei), to the bovine enzyme and to recombinant human DHFR. However, binding is sufficiently tight, especially in the ternary complexes with NADPH, for recording of the EPR spectra of the bound ligands. The spectra indicate that when these inhibitors are bound to the enzyme the TEMPO group is highly immobilized with correlation time, tau c, 4-20ns. Inhibitors that have the reporter group attached to the glutamate moiety of methotrexate bind to all four DHFRs more tightly than the inhibitors with shorter side chains by factors of up to 10(6). However, in most complexes formed by the inhibitors with longer side chains immobilization of the TEMPO group is slight (tau c 0.2-4 ns). These results are in general agreement with predictions from X-ray crystallographic results including thermal factors but there are some unanticipated differences between some results for bacterial and eukaryotic enzymes. Three of the splin-labeled inhibitors would provide good probes for distance measurements in and around the active site of mammalian DHFR.     

Regulation of serine protease activity by an engineered metal switch

A recombinant trypsin was designed whose catalytic activity can be regulated by varying the concentration of Cu2+ in solution. Substitution of Arg-96 with a His in rat trypsin (trypsin R96H) places a new imidazole group on the surface of the enzyme near the essential active-site His-57. The unique spatial orientation of these His side chains results in the formation of a stable, metal-binding site that chelates divalent first-row transition-metal ions. Occupancy of this site by a metal ion prevents the imidazole group of His-57 from participating as a general base in catalysis. As a consequence, the primary effect of the transition metal ion is to inhibit the esterase and amidase activities of trypsin R96H. The apparent Ki for this inhibition is in the micromolar range for copper, nickel, and zinc, the tightest binding being to Cu2+ at 21 microM. Trypsin R96H activity can be fully restored by removing the bound Cu2+ ion with EDTA. Multiple cycles of inhibition by Cu2+ ions and reactivation by EDTA demonstrate that reversible regulatory control has been introduced into the enzyme. These results describe a novel mode of inhibition of serine protease activity that may also prove applicable to other proteins.     

Site-specific labeling of cytoplasmic peptide:N-glycanase by N,N'-diacetylchitobiose-related compounds

Peptide:N-glycanase (PNGase) is the deglycosylating enzyme, which releases N-linked glycan chains from N-linked glycopeptides and glycoproteins. Recent studies have revealed that the cytoplasmic PNGase is involved in the degradation of misfolded/unassembled glycoproteins. This enzyme has a Cys, His, and Asp catalytic triad, which is required for its enzymatic activity and can be inhibited by "free" N-linked glycans. These observations prompted us to investigate the possible use of haloacetamidyl derivatives of N-glycans as potent inhibitors and labeling reagents of this enzyme. Using a cytoplasmic PNGase from budding yeast (Png1), Man9GlcNAc2-iodoacetoamide was shown to be a strong inhibitor of this enzyme. The inhibition was found to be through covalent binding of the carbohydrate to a single Cys residue on Png1, and the binding was highly selective. The mutant enzyme in which Cys191 of the catalytic triad was changed to Ala did not bind to the carbohydrate probe, suggesting that the catalytic Cys is the binding site for this compound. Precise determination of the carbohydrate attachment site by mass spectrometry clearly identified Cys191 as the site of covalent attachment. Molecular modeling of N,N'-diacetylchitobiose (chitobiose) binding to the protein suggests that the carbohydrate binding site is distinct from but adjacent to that of Z-VAD-fmk, a peptide-based inhibitor of this enzyme. These results suggest that cytoplasmic PNGase has a separate binding site for chitobiose and other carbohydrates, and haloacetamide derivatives can irreversibly inhibit that catalytic Cys in a highly specific manner.     

Reversible S-nitrosation and inhibition of HIV-1 protease.

Nitric oxide (*NO) is a short-lived free radical with many functions including vasoregulation, synaptic plasticity, and immune modulation and has recently been associated with AIDS pathology. Various pathophysiological conditions, such as viral infection, trigger inducible nitric oxide synthase (iNOS) to synthesize NO in the cell. NO-derived species can react with thiols of proteins and form nitrosothiol adducts. HIV-1 protease (HIV-PR) contains two cysteine residues, Cys67 and Cys95, which are believed to serve a regulatory function. We have found that HIV-PR is inactivated by nitric oxide produced in vitro by NO donors and by iNOS. Sodium nitroprusside inhibited HIV-PR by 70%, and S-nitroso-N-acetylpenicillamine completely inhibited the enzyme. Furthermore, iNOS generated sufficient NO to inhibit HIV-PR activity by almost 90%. This inactivation was reversed by the addition of reducing agents. Treatment of HIV-PR with NO donors and ritonavir (a competitive peptide inhibitor) indicates that NO exerts its effect through a site independent of the active site of HIV-PR. Using electrospray ionization mass spectrometry, we found that NO forms S-nitrosothiols on Cys67 and Cys95 of HIV-PR which directly correlate with a loss of activity. These data indicate that NO may suppress HIV-1 replication by directly inhibiting HIV-PR.     

L-aspartase from Escherichia coli: substrate specificity and role of divalent metal ions

The enzyme L-aspartase from Escherichia coli has an absolute specificity for its amino acid substrate. An examination of a wide range of structural analogues of L-aspartic acid did not uncover any alternate substrates for this enzyme. A large number of competitive inhibitors of the enzyme have been characterized, with inhibition constants ranging over 2 orders of magnitude. A divalent metal ion is required for enzyme activity above pH 7, and this requirement is met by many transition and alkali earth metals. The binding stoichiometry has been established to be one metal ion bound per subunit. Paramagnetic relaxation studies have shown that the divalent metal ion binds at the recently discovered activator site on L-aspartase and not at the enzyme active site. Enzyme activators are bound within 5 A of the enzyme-bound divalent metal ion. The activator site is remote from the active site of the enzyme, since the relaxation of inhibitors that bind at the active site is not affected by paramagnetic metal ions bound at the activator site.     

Kinetic and physical properties of Co2+ enolase

The activation of yeast enolase by cobaltous ion in 0.1 M KCl is characterized by an activation constant of 1 microM and an inhibition constant of 18 microM. Measurements of binding of Co2+ to the apoenzyme show that a maximum of four Co2+ ions are bound per dimer in the presence or absence of substrate although binding is far tighter in the presence of substrate. Ultraviolet spectral titrations show evidence for a conformational change due exclusively to the binding of the first two ions of Co2+. Both visible and EPR spectra confirm that the environment of the first pair of cobalt ions ("conformational sites") is markedly different from that of the second pair in the "catalytic" sites. Cobalt at the conformational site appears to be a tetragonally distorted octahedral complex while the second pair of metal ions appears to be in a more regular tetrahedral symmetry. Addition of either Mg2+ or substrate to the enzyme with only one pair of cobalt ions per dimer causes striking changes in the metal ion environment. The conformational metal sites appear sufficiently shielded from solvent to be inaccessible to oxidation by H2O2, in contrast to the second pair of cobaltous ions whose ready oxidation by H2O2 inactivates the enzyme. Comparison of kinetic and binding data suggests that only one site of the dimeric enzyme can be active, since activity requires more than two metals bound per dimer and inactivation results from the binding of the fourth ion per dimer.     

Lanthanide spectroscopic studies of the dinuclear and Mg(II)-dependent PvuII restriction endonuclease

Type II restriction enzymes are homodimeric systems that bind four to eight base pair palindromic recognition sequences of DNA and catalyze metal ion-dependent phosphodiester cleavage. While Mg(II) is required for cleavage in these enzymes, in some systems Ca(II) promotes avid substrate binding and sequence discrimination. These properties make them useful model systems for understanding the roles of alkaline earth metal ions in nucleic acid processing. We have previously shown that two Ca(II) ions stimulate DNA binding by PvuII endonuclease and that the trivalent lanthanide ions Tb(III) and Eu(III) support subnanomolar DNA binding in this system. Here we capitalize on this behavior, employing a unique combination of luminescence spectroscopy and DNA binding assays to characterize Ln(III) binding behavior by this enzyme. Upon excitation of tyrosine residues, the emissions of both Tb(III) and Eu(III) are enhanced severalfold. This enhancement is reduced by the addition of a large excess of Ca(II), indicating that these ions bind in the active site. Poor enhancements and affinities in the presence of the active site variant E68A indicate that Glu68 is an important Ln(III) ligand, similar to that observed with Ca(II), Mg(II), and Mn(II). At low micromolar Eu(III) concentrations in the presence of enzyme (10-20 microM), Eu(III) excitation (7)F(0) --> (5)D(0) spectra yield one dominant peak at 579.2 nm. A second, smaller peak at 579.4 nm is apparent at high Eu(III) concentrations (150 microM). Titration data for both Tb(III) and Eu(III) fit well to a two-site model featuring a strong site (K(d) = 1-3 microM) and a much weaker site (K(d) approximately 100-200 microM). Experiments with the E68A variant indicate that the Glu68 side chain is not required for the binding of this second Ln(III) equivalent; however, the dramatic increase in DNA binding affinity around 100 microM Ln(III) for the wild-type enzyme and metal-enhanced substrate affinity for E68A are consistent with functional relevance for this weaker site. This discrimination of sites should make it possible to use lanthanide substitution and lanthanide spectroscopy to probe individual metal ion binding sites, thus adding an important tool to the study of restriction enzyme structure and function.     

Interaction of metal ions with carboxylic and carboxamide groups in protein structures

An analysis of the geometry of metal binding by carboxylic and carboxamide groups in proteins is presented. Most of the ligands are from aspartic and glutamic acid side chains. Water molecules bound to carboxylate anions are known to interact with oxygen lone-pairs. However, metal ions are also found to approach the carboxylate group along the C-O direction. More metal ions are found to be along the syn than the anti lone-pair direction. This seems to be the result of the stability of the five-membered ring that is formed by the carboxylate anion hydrogen bonded to a ligand water molecule and the metal ion in the syn position. Ligand residues are usually from the helix, turn or regions with no regular secondary structure. Because of the steric interactions associated with bringing all the ligands around a metal center, a calcium ion can bind only near the ends of a helix; a metal, like zinc, with a low coordination number, can bind anywhere in the helix. Based on the analysis of the positions of water molecules in the metal coordination sphere, the sequence of the EF hand (a calcium-binding structure) is discussed.     

Binding of metal cyanide complexes to bovine liver rhodanese in the crystalline state

Bovine liver rhodanese, which catalyzes the transfer of sulfur atoms between a variety of sulfur donor and sulfur acceptor substrates, is inhibited by metal cyanide complexes [Volini, M., Van Sweringen, B., & Chen, F.-Sh. (1978) Arch. Biochem. Biophys. 191, 205-215]. Crystallographic studies are described which reveal the binding mode of four different metal cyanides to bovine liver rhodanese: Na[Au(CN2], K2[Pt(CN)4], K2[Ni(CN)4], and K2[Zn(CN)4]. It appears that these complexes bind at one common site at the entrance of the active site pocket, interacting with the positively charged side chains of Arg-186 and Lys-249. This observation explains the inhibition of rhodanese by this class of compounds. For the platinum and nickel cyanide complexes virtually no other binding sites are observed. The gold complex binds, however, to three additional cysteine residues, thereby also displacing the extra sulfur atom which was bound to the essential Cys-247 in the sulfur-rhodanese complex. The zinc complex binds to completely different additional sites and forms complexes with the side chains of Asp-101 and His-203. Possible reasons for these different binding modes are discussed in terms of the preference for "hard" and "soft" ligands of these four metal ions.     

Structure–function analysis of cytochromes P450 2B

In the last 4 years, breakthroughs were made in the field of P450 2B (CYP2B) structure–function through determination of one ligand-free and two inhibitor-bound X-ray crystal structures of CYP2B4, which revealed many of the structural features required for binding ligands of different size and shape. Large conformational changes of several plastic regions of CYP2B4 can dramatically reshape the active site of the enzyme to fit the size and shape of the bound ligand without perturbing the overall P450 fold. Solution biophysical studies using isothermal titration calorimetry (ITC) have revealed the large difference in the thermodynamic parameters of CYP2B4 in binding inhibitors of different ring chemistry and side chains. Other studies have revealed that the effects of site-specific mutations on steady-state kinetic parameters and mechanism-based inactivation are often substrate dependent. These findings agree with the structural data that the enzymes adopt different conformations to bind various ligands. Thus, the substrate specificity of an individual enzyme is determined not only by active site residues but also non-active site residues that modulate conformational changes that are important for substrate access and rearrangement of the active site to accommodate the bound substrate.     

Structure-function analysis of cytochromes P450 2B

In the last 4 years, breakthroughs were made in the field of P450 2B (CYP2B) structure-function through determination of one ligand-free and two inhibitor-bound X-ray crystal structures of CYP2B4, which revealed many of the structural features required for binding ligands of different size and shape. Large conformational changes of several plastic regions of CYP2B4 can dramatically reshape the active site of the enzyme to fit the size and shape of the bound ligand without perturbing the overall P450 fold. Solution biophysical studies using isothermal titration calorimetry (ITC) have revealed the large difference in the thermodynamic parameters of CYP2B4 in binding inhibitors of different ring chemistry and side chains. Other studies have revealed that the effects of site-specific mutations on steady-state kinetic parameters and mechanism-based inactivation are often substrate dependent. These findings agree with the structural data that the enzymes adopt different conformations to bind various ligands. Thus, the substrate specificity of an individual enzyme is determined not only by active site residues but also non-active site residues that modulate conformational changes that are important for substrate access and rearrangement of the active site to accommodate the bound substrate.     

Endogenous effectors of human liver glycogen phosphorylase modulate effects of indole-site inhibitors

Phosphorylase is regulated by a number of small-molecular-weight effectors that bind to three sites on the enzyme. Recently, a fourth site referred to as the indole-inhibitor site has been identified. Synthetic compounds bind to the site and inhibit activity. However, the effects of these compounds in the presence of other endogenous effectors are unknown. We have determined the effects of four indole derivative glycogen phosphorylase inhibitors (GPI) on recombinant human liver glycogen phosphorylase a activity. The GPIs tested were all potent inhibitors. However, the endogenous inhibitors (glucose, ADP, ATP, fructose 1-phosphate, glucose 6-phosphate, UDP-glucose) and the activator (AMP) markedly reduced the inhibitory effect of GPIs. Consistent with these in vitro findings, the IC50 for the inhibition of glycogenolysis in cells and the liver drug concentration associated with glucose-lowering activity in diabetic ob/ob mice in vivo were also significantly higher than those determined in in vitro enzyme assays. The inhibitory effect of indole-site effectors is modulated by endogenous small-molecular-weight effectors of phosphorylase a activity. However, at higher concentrations (10-30 microM), the GPI effect was dominant and resulted in inhibition of phosphorylase a activity irrespective of the presence or absence of the other modulators of the enzyme.     

Purification and properties of glucose 6-phosphate dehydrogenase from Aspergillus aculeatus

Glucose 6-phosphate dehydrogenase (EC 1.1.1.49) was purified from Aspergillus aculeatus, a filamentous fungus previously isolated from infected tongue of a patient. The enzyme, apparently homogeneous, had a specific activity of 220 units mg(-1), a molecular weight of 105,000 +/- 5,000 Dal by gel filtration and subunit size of 52,000 +/- 1,100 Dal by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The substrate specificity was extremely strict, with glucose 6-phosphate (G6P) being oxidized by nicotinamide adenine dinucleotide phosphate (NADP) only. At assay pH of 7.5, the enzyme had K(m) values of 6 microM and 75 microM for NADP and G6P respectively. The k(cat) was 83 s(-1). Steady-state kinetics at pH 7.5 produced converging linear Lineweaver-Burk plots as expected for ternary-complex mechanism. The patterns of product and dead-end inhibition suggested that the enzyme can bind NADP and G6P separately to form a binary complex, indicating a random-order mechanism. The enzyme was irreversibly inactivated by heat in a linear fashion, with G6P providing a degree of protection. Phosphoenolpyruvate (PEP), adenosinetriphosphate (ATP), and fructose 6-phosphate (F6P), in decreasing order, are effective inhibitors. Zinc and Cobalt ions were effective inhibitors although cobalt ion was more potent; the two divalent metals were competitive inhibitors with respect to G6P, with Ki values of 6.6 microM and 4.7 microM respectively. It is proposed that inhibition by divalent metal ions, at low NADPH /NADP ratio, is another means of controlling pentosephosphate pathway.     

X-ray absorption spectroscopy structural investigation of early intermediates in the mechanism of DNA repair by human ABH2

Human ABH2 repairs DNA lesions by using an Fe(II)- and αKG-dependent oxidative demethylation mechanism. The structure of the active site features the facial triad of protein ligands consisting of the side chains of two histidine residues and one aspartate residue that is common to many non-heme Fe(II) oxygenases. X-ray absorption spectroscopy (XAS) of metallated (Fe and Ni) samples of ABH2 was used to investigate the mechanism of ABH2 and its inhibition by Ni(II) ions. The data are consistent with a sequential mechanism that features a five-coordinate metal center in the presence and absence of the α-ketoglutarate cofactor. This aspect is not altered in the Ni(II)-substituted enzyme, and both metals are shown to bind the cofactor. When the substrate is bound to the native Fe(II) complex with α-ketoglutarate bound, a five-coordinate Fe(II) center is retained that features an open coordination position for O(2) binding. However, in the case of the Ni(II)-substituted enzyme, the complex that forms in the presence of the cofactor and substrate is six-coordinate and, therefore, features no open coordination site for oxygen activation at the metal.     

Synthesis and biological evaluation of novel flavonoid derivatives as dual binding acetylcholinesterase inhibitors

A new series of flavonoid derivatives have been designed, synthesised and evaluated as acetylcholinesterase inhibitors that could bind simultaneously to the peripheral and catalytic sites of the enzyme. Among them, fifteen derivatives were found to inhibit the enzyme in the micromolar range and isoflavone derivatives possessed more potent inhibitory activity than other flavonoid derivatives. The best compound 9a had its inhibitory activity (IC(50) = 0.093 microM) in the same range as the reference compound, donepezil (IC(50) = 0.025 microM). Preliminary structure-activity relationships and a molecular modeling study for 9a have revealed that the isoflavone moiety plays a key role in the interaction of this series of derivatives with AChE by acting as an anchor in its peripheral anionic site.     

Effects of cadmium on manganese peroxidase competitive inhibition of MnII oxidation and thermal stabilization of the enzyme.

Inhibition of manganese peroxidase by cadmium was studied under steady-state and transient-state kinetic conditions. CdII is a reversible competitive inhibitor of MnII in the steady state with Ki approximately 10 microM. CdII also inhibits enzyme-generated MnIII-chelate-mediated oxidation of 2,6-dimethoxyphenol with Ki approximately 4 microM. CdII does not inhibit direct oxidation of phenols such as 2,6-dimethoxyphenol or guaiacol (2-methoxyphenol) in the absence of MnII. CdII alters the heme Soret on binding manganese peroxidase and exhibits a Kd approximately 8 microM, similar to Mn (Kd approximately 10 microM). Under transient-state conditions, CdII inhibits reduction of compound I and compound II by MnII at pH 4.5. However, CdII does not inhibit formation of compound I nor does it inhibit reduction of the enzyme intermediates by phenols in the absence of MnII. Kinetic analysis suggests that CdII binds at the MnII-binding site, preventing oxidation of MnII, but does not impair oxidation of substrates, such as phenols, which do not bind at the MnII-binding site. Finally, at pH 4.5 and 55 degrees C, MnII and CdII both protect manganese peroxidase from thermal denaturation more efficiently than CaII, extending the half-life of the enzyme by more than twofold. Furthermore, the combination of half MnII and half CdII nearly quadruples the enzyme half-life over either metal alone or either metal in combination with CaII.