Additional Active Site Research Articles

Role of oxygen vacancies in the basicity of ZnO: From the model methylbutynol conversion to the ethanol transformation application

The parameters governing the basic reactivity of kadox and ex-carbonate zinc oxide samples towards alcohol conversion were investigated varying the atmosphere of pre-treatment for a model reaction, the methylbutynol (MBOH) conversion, and the ethanol conversion reaction, a transformation of potential higher industrial impact. The catalytic properties of ZnO can thus be tuned: enhanced activities were measured after a pre-treatment performed under inert gas as compared to oxidative conditions. If no significant variation of residual hydroxylation nor carbonation can account for this behaviour, a quantitative correlation between the variation of MBOH conversion induced by the different atmosphere of pre-treatment with that of the concentration in oxygen vacancies characterized by in situ EPR found, showing that oxygen vacancies play a key role towards basic reactivity. In fact, the electronic density of the oxygen of the active acid/base pair is directly influenced by the electron release or capture associated to the formation or filling up of oxygen vacancies, respectively. On the kadox samples, the presence of an additional weaker active site is responsible for the residual activity of kadox sample in the absence of oxygen vacancies. The existence and location on specific crystallographic faces of these sites are discussed in relation with the different morphologies of the two samples and with their different affinity with CO2. Even if the occurrence of oxygen vacancies still governs the conversion level for ethanol transformation, the reactivity inhibition after oxidative pre-treatment is less pronounced due to the higher reaction temperature that limits the efficiency of oxygen vacancies filling-up under oxidative atmospheres. The nature of active sites leading to the formation of acetaldehyde and ethylene is also discussed.

Binding and inactivation mechanism of a humanized fatty acid amide hydrolase by alpha-ketoheterocycle inhibitors revealed from cocrystal structures.

The cocrystal X-ray structures of two isomeric alpha-ketooxazole inhibitors (1 (OL-135) and 2) bound to fatty acid amide hydrolase (FAAH), a key enzymatic regulator of endocannabinoid signaling, are disclosed. The active site catalytic Ser241 is covalently bound to the inhibitors' electrophilic carbonyl groups, providing the first structures of FAAH bound to an inhibitor as a deprotonated hemiketal mimicking the enzymatic tetrahedral intermediate. The work also offers a detailed view of the oxyanion hole and an exceptional "in-action" depiction of the unusual Ser-Ser-Lys catalytic triad. These structures capture the first picture of inhibitors that span the active site into the cytosolic port providing new insights that help to explain FAAH's interaction with substrate leaving groups and their role in modulating inhibitor potency and selectivity. The role for the activating central heterocycle is clearly defined and distinguished from that observed in prior applications with serine proteases, reconciling the large electronic effect of attached substituents found unique to this class of inhibitors with FAAH. Additional striking active site flexibility is seen upon binding of the inhibitors, providing insights into the existence of a now well-defined membrane access channel with the disappearance of a spatially independent portion of the acyl chain-binding pocket. Finally, comparison of the structures of OL-135 (1) and its isomer 2 indicates that they bind identically to FAAH, albeit with reversed orientations of the central activating heterocycle, revealing that the terminal 2-pyridyl substituent and the acyl chain phenyl group provide key anchoring interactions and confirming the distinguishing role of the activating oxazole.

Structure of the Human Lung Cytochrome P450 2A13

The human lung cytochrome P450 2A13 (CYP2A13) activates the nicotine-derived procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) into DNA-altering compounds that cause lung cancer. Another cytochrome P450, CYP2A6, is also present in human lung, but at much lower levels. Although these two enzymes are 93.5% identical, CYP2A13 metabolizes NNK with much lower K(m) values than does CYP2A6. To investigate the structural differences between these two enzymes the structure of CYP2A13 was determined to 2.35A by x-ray crystallography and compared with structures of CYP2A6. As expected, the overall CYP2A13 and CYP2A6 structures are very similar with an average root mean square deviation of 0.5A for the Calpha atoms. Like CYP2A6, the CYP2A13 active site cavity is small and highly hydrophobic with a cluster of Phe residues composing the active site roof. Active site residue Asn(297) is positioned to hydrogen bond with an adventitious ligand, identified as indole. Amino acid differences between CYP2A6 and CYP2A13 at positions 117, 300, 301, and 208 relate to different orientations of the ligand plane in the two protein structures and may underlie the significant variations observed in binding and catalysis of many CYP2A ligands. In addition, docking studies suggest that residues 365 and 366 may also contribute to differences in NNK metabolism.

Exon grafting yields a “two active-site” lysozyme

The design of enzymes with enhanced stability and activity has long been a goal in protein engineering. We report a strategy to engineer an additional active site for human lysozyme, grafted the entire human lysozyme exon 2, which encodes the catalytically competent domain, into the gene at a position corresponding to an exposed loop region in the translated protein. Exon 2 grafting created a novel lysozyme with twice the activity of the wild type enzyme, equal activity came from each of the two active sites. We dissected the contributions of each active site using site-directed mutagenesis of the catalytic doublets of (E35A/D53A), circular dichroism, fluorescence spectra, and molecular modeling. Temperature and pH stability of the “two active-site” enzyme were similar to those of wild-type lysozyme. Thus, we provide a novel strategy for engineering the active site of enzymes.

Introduction of a pi-pi interaction at the active site of a cupredoxin: characterization of the Met16Phe Pseudoazurin mutant.

The Met16Phe mutant of the type 1 copper protein pseudoazurin (PACu), in which a phenyl ring is introduced close to the imidazole moiety of the His81 ligand, has been characterized. NMR studies indicate that the introduced phenyl ring is parallel to the imidazole group of His81. The mutation has a subtle effect on the position of the two S(Cys)-->Cu(II) ligand-to-metal charge transfer bands in the visible spectrum of PACu(II) and a more significant influence on their intensities resulting in a A(459)/A(598) ratio of 0.31 for Met16Phe as compared to a A(453)/A(594) ratio of 0.43 for wild-type PACu(II) at pH 8. The electron paramagnetic resonance spectrum of the Met16Phe variant is more axial than that of the wild-type protein, and the resonance Raman spectrum of the mutant exhibits subtle differences. A C(gamma)H proton of Met86 exhibits a much smaller hyperfine shift in the paramagnetic (1)H NMR spectrum of Met16Phe PACu(II) as compared to its position in the wild-type protein, which indicates a weaker axial Cu-S(Met86) interaction in the mutant. The Met16Phe mutation results in an approximately 60 mV increase in the reduction potential of PACu. The pK(a) value of the ligand His81 decreases from 4.9 in wild-type PACu(I) to 4.5 in Met16Phe PACu(I) indicating that the pi-pi contact with Phe16 stabilizes the Cu-N(His81) interaction. The Met16Phe variant of PACu has a self-exchange rate constant at pH 7.6 (25 degrees C) of 9.8 x 10(3) M(-)(1) s(-)(1) as compared to the considerably smaller value of 3.7 x 10(3) M(-)(1) s(-)(1) for the wild-type protein under identical conditions. The enhanced electron transfer reactivity of Met16Phe PACu is a consequence of a lower reorganization energy due to additional active site rigidity caused by the pi-pi interaction between His81 and the introduced phenyl ring.

Expression of Moloney murine leukemia virus RNase H rescues the growth defect of an Escherichia coli mutant.

A 157-amino-acid fragment of Moloney murine leukemia virus reverse transcriptase encoding RNase H is shown to rescue the growth-defective phenotype of an Escherichia coli mutant. In vitro assays of the recombinant wild-type protein purified from the conditionally defective mutant confirm that it is catalytically active. Mutagenesis of one of the presumptive RNase H-catalytic residues results in production of a protein variant incapable of rescue and which lacks activity in vitro. Analyses of additional active site mutants demonstrate that their encoded variant proteins lack robust activity yet are able to rescue the bacterial mutant. These results suggest that genetic complementation may be useful for in vivo screening of mutant viral RNase H gene fragments and in evaluating their function under conditions that more closely mimic physiological conditions. The rescue system may also be useful in verifying the functional outcomes of mutations based on protein structural predictions and modeling.

Calpain, an upstream regulator of thymocyte apoptosis.

Apoptosis is the common phenotype of programmed or physiologic cell death, the process used to remove excess or defunct cells during normal tissue maintenance. One of the most studied cell types with respect to apoptosis is the immature T cell from the thymus, which activates its death program in response to an enormous variety of agents. Previously, our group implicated the calcium-dependent cytosolic protease calpain as a participant in thymocyte apoptosis initiated by glucocorticoids or irradiation. We found that the calpain inhibitors N-acetyl-leu-leu-norleucinal (calpain inhibitor I) and carbenzoxy-val-phe-H (MDL 28,170) prevented dexamethasone-induced apoptosis of thymocytes; in this study, we show that two additional calpain active site inhibitors, L-3-carboxy-trans-2,3-epoxypropionyl-leu-amido-(4-guanidinio )butane ethyl ester (E64d) and carbenzoxy-leu-leu-tyr-CHN2 (ZLLY-CHN2), also prevent apoptosis in this model. Three compounds that inhibit lysosomal cysteine proteases, carbenzoxy-tyr-ala-CHN2 (ZYA-CHN2), ammonium chloride, and chloroquine, do not block apoptosis, indicating that the effect of the calpain inhibitors is not due to cross-inhibition of lysosomal proteases. In addition, I-benzyl-CH=C(SH)COOH (PD150606), a calpain inhibitor directed toward the calcium binding sites of calpain, also prevents apoptosis. Calpain is necessary for other models of programmed cell death that require new gene expression (induction models), those following treatment of thymocytes with the calcium ionophore A23187, ionomycin, or forskolin. However, two models of thymocyte apoptosis that do not require new gene expression (transduction models), those triggered by heat shock and by valinomycin, are calpain independent, as is calcium-triggered DNA fragmentation in isolated thymocyte nuclei. These experiments suggest an upstream regulatory role for calpain in a pathway to thymocyte apoptosis common to several inducers.

Structure of recombinant ricin A chain at 2.3 A.

The plant cytotoxin ricin is a heterodimer with a cell surface binding (B) chain and an enzymatically active A chain (RTA) known to act as a specific N-glycosidase. RTA must be separated from B chain to attack rRNA. The X-ray structure of ricin has been solved recently; here we report the structure of the isolated A chain expressed from a clone in Escherichia coli. This structure of wild-type rRTA has and will continue to serve as the parent compound for difference Fouriers used to assess the structure of site-directed mutants designed to analyze the mechanism of this medically and commercially important toxin. The structure of the recombinant protein, rRTA, is virtually identical to that seen previously for A chain in the heterodimeric toxin. Some minor conformational changes due to interactions with B chain and to crystal packing differences are described. Perhaps the most significant difference is the presence in rRTA of an additional active site water. This molecule is positioned to act as the ultimate nucleophile in the depurination reaction mechanism proposed by Monzingo and Robertus (1992, J. Mol. Biol. 227, 1136-1145).

Fluorescent properties of the Escherichia coli D-xylose isomerase active site.

The consequences of active site mutations of the Escherichia coli D-xylose isomerase (E.C. 5.3.1.5) on substrate binding were examined by fluorescence spectroscopy. Site-directed mutagenesis of conserved tryptophan residues in the E. coli enzyme (Trp49 and Trp188) reveals that fluorescence quenching of these residues occurs during the binding of xylose by the wild-type enzyme. The fluorescent properties of additional active site substitutions at His101 were also examined. Substitutions of His101 which inactivate the enzyme were shown to have altered spectral characteristics, which preclude detection of substrate binding. In the case of H101S, a mutant protein with measurable isomerizing activity, substrate binding with novel fluorescent properties was observed, possibly the bound pyranose form of xylose under steady-state conditions.

Down-regulation of fast-twitch skeletal muscle fiber with cardiac troponin-C and recombinant mutants Structure/function studies with site-directed mutagenesis

Structure/function relationships in troponin C are studied with vertebrate fast-twitch fibers by exchanging the skeletal troponin C with two bacterially synthesized recombinant proteins designed by site-directed mutagenesis of cardiac troponin C. One mutant (CBM1) contained an additional active site, by deleting Val-28 and converting Leu-29, Gly-30, Ala-31 and Glu-32 to Asp, Ala, Asp and Gly, respectively, in the normally inactive trigger site 1 in the N-terminus. In another mutant (CBM2A), the normally active site 2 was inactivated by conversion of Asp-65 to Ala. The fibers were found to be down-regulated with recombinant cardiac troponin C (CTnC3), as with tissue-cardiac-troponin-C. With mutants, in one case (CBM1) the regulation was unmodified despite Ca2+ coordination by all sites. In contrast, regulation was found to be completely blocked with the mutant (CBM2A) where both trigger sites were inactive. The results provide the first indication that structural specification of the entire EF-hand motif of site 1, and not just Ca2+ coordination, is needed to operate fully the Ca2+ switch in fast-twitch fibers.