Putative Transition-state Analogue Research Articles

A plant pentatricopeptide repeat protein with a DYW-deaminase domain is sufficient for catalyzing C-to-U RNA editing in vitro

Pentatricopeptide repeat (PPR) proteins with C-terminal DYW domains are present in organisms that undergo C-to-U editing of organelle RNA transcripts. PPR domains act as specificity factors through electrostatic interactions between a pair of polar residues and the nitrogenous bases of an RNA target. DYW-deaminase domains act as the editing enzyme. Two moss (Physcomitrella patens) PPR proteins containing DYW-deaminase domains, PPR65 and PPR56, can convert Cs to Us in cognate, exogenous RNA targets co-expressed in Escherichia coli We show here that purified, recombinant PPR65 exhibits robust editase activity on synthetic RNAs containing cognate, mitochondrial PpccmFC sequences in vitro, indicating that a PPR protein with a DYW domain is solely sufficient for catalyzing C-to-U RNA editing in vitro Monomeric fractions possessed the highest conversion efficiency, and oligomeric fractions had reduced activity. Inductively coupled plasma (ICP)-MS analysis indicated a stoichiometry of two zinc ions per highly active PPR65 monomer. Editing activity was sensitive to addition of zinc acetate or the zinc chelators 1,10-o-phenanthroline and EDTA. Addition of ATP or nonhydrolyzable nucleotide analogs stimulated PPR65-catalyzed RNA-editing activity on PpccmFC substrates, indicating potential allosteric regulation of PPR65 by ATP. Unlike for bacterial cytidine deaminase, addition of two putative transition-state analogs, zebularine and tetrahydrouridine, failed to disrupt RNA-editing activity. RNA oligonucleotides with a single incorporated zebularine also did not disrupt editing in vitro, suggesting that PPR65 cannot bind modified bases due to differences in the structure of the active site compared with other zinc-dependent nucleotide deaminases.

Evaluating hydrophobic galactonoamidines as transition state analogs for enzymatic β-galactoside hydrolysis

A spectroscopic examination of six galactonoamidines with inhibition constants and efficacy in the low nanomolar concentration range (Ki = 6–11 nM, IC50 = 12–36 nM) suggested only two of them as putative transition state analogs for the hydrolysis of β-galactosides by β-galactosidase (A. oryzae). A rationale for the experimental results was elaborated using docking and molecular dynamics studies. An analysis of the combined observations reveals several common factors of the compounds suggested as transition state analogs (TSAs): the putative TSAs have a similar orientation in the active site; show conserved positioning of the glycon; display a large number of H-bond interactions toward the catalytically active amino acid residues via their glycon; and exhibit hydrophobic interactions at the outer rim of the active site with small changes of the position and orientation of their respective aglycons.

Evaluating N-benzylgalactonoamidines as putative transition state analogs for β-galactoside hydrolysis

Experimental evidence is provided for p-methylbenzyl-D-galactonoamidine to function as a true transition state analog for the enzymatic hydrolysis of aryl-β-D-galactopyranosides by β-galactosidase (A. oryzae). The compound exhibits inhibition constants in the low nanomolar concentration range (12-56 nM) for a selection of substrates. Along these lines, a streamlined synthetic method based on phase-transfer catalysis was optimized to afford the required variety of new aryl-β-D-galactopyranosides. Last, the stability of the galactonoamidines under the assay conditions was confirmed.

Detection of nucleotide- and F-actin-induced movements in the switch II helix of the skeletal myosin using its differential oxidative cleavage mediated by an iron-EDTA complex disulfide-linked to the strong actin binding site.

We have synthesized the mixed disulfide, S-(2-nitro-5-thiobenzoic acid) cysteaminyl-EDTA, using a rapid procedure and water-soluble chemistry. Its disulfide-thiol exchange reaction with rabbit myosin subfragment-1 (S-1), analyzed by spectrophotometry, ATPase assays, and peptide mapping, led to the incorporation of the cysteaminyl-EDTA group into only Cys 540 on the heavy chain and into the unique cysteine on the alkali light chains. The former thiol, residing in the strong actin binding site, reacted at a much faster rate with a concomitant 3-fold decrease in the V(max) for acto-S-1 ATPase but without change in the essential enzymatic functions of S-1. Upon chelation of Fe(3+) ions to the Cys 540-bound EDTA and incubation of the S-1 derivative-Fe complex with ascorbic acid at pH 7.5, the 95 kDa heavy chain underwent a conformation-dependent, single-cut oxidative fragmentation within 5-15 A of Cys 540. Three pairs of fragments were formed which, after specific fluorescent labeling and SDS-PAGE, could be positioned along the heavy chain sequence as 68 kDa-26 kDa, 62 kDa-32 kDa, and 54 kDa-40 kDa. Densitometric measurements revealed that the yield of the 54 kDa-40 kDa pair of bands, but not that for the two other pairs, was very sensitive to S-1 binding to nucleotides or phosphate analogues as well as to F-actin. In binary complexes, all the former ligands specifically lowered the yield to 40% of S-1 alone, roughly in the following order: ADP = AMP-PNP > ATP = ADP.AlF(4) > ADP.BeF(x)() > PP(i). By contrast, rigor binding to F-actin increased the yield to 130%. In the ternary acto-S-1-ADP complex, the yield was again reduced to 80%, and it fell to 25% in acto-S-1-ADP.AlF(4), the putative transition state analogue complex of the acto-S-1 ATPase. These different quantitative changes reflect distinct ligand-induced conformations of the secondary structure element whose scission generates the 54 kDa-40 kDa species. According to the S-1 crystal structure, this element could be unambiguously assigned to the switch II helix (residues 475-507) whose N-terminus lies 14.2 A from Cys 540 and would include the ligand-responsive cleavage site. This motif is thought to be crucial for the transmission of sub-nanometer structural changes at the ATPase site to both the actin site and the lever arm domain during energy transduction. Our study illustrates this novel, actin site-specific chemical proteolysis of S-1 as a direct probe of the switch II helix conformational transitions in solution most likely associated with the skeletal cross-bridge cycle.

Systematic study of the six cysteines of the E1 subunit of the pyruvate dehydrogenase multienzyme complex from Escherichia coli: none is essential for activity.

Variants of the Escherichia coli 1-lip pyruvate dehydrogenase multienzyme complex (1-lip PDHc) with the C259N and C259S substitutions in the putative thiamin diphosphate-(ThDP-) binding motif of the pyruvate dehydrogenase component (E1, EC 1.2.4.1) were characterized. Single substitutions were made at the five remaining cysteines of the E1 component, creating the C120A, C575A, C610A, C654A, and C770S variants to test the hypothesis that the activity loss that accompanies exposure of the enzyme to fluoropyruvate, bromopyruvate, and 2-oxo-3-butynoic acid is the result of the modification of approximately one cysteine residue per E1 monomer. Surprisingly, all single cysteine E1 variants could be reconstituted with E2-E3 subcomplex and showed PDHc activity ranging from 74% to 96% that of the parental enzyme. The specific activities of C259N and C259S variants of 1-lip PDHc were 58% and 27% relative to that of the parental 1-lip PDHc. All five single cysteine E1 variants, along with the C259N and C259S variants of 1-lip PDHc, could also (1) be inactivated with fluoropyruvate and 2-oxo-3-butynoic acid, (2) were subject to inactivation by the monoclonal antibody 18A9 reported from one of our laboratories, and (3) were subject to regulation by pyruvate and acetyl-CoA. It was therefore concluded that none of the six cysteine residues is essential for the activity of the E1 component or of the complex. When tested with the putative transition-state analogue, thiamin 2-thiothiazolone diphosphate, all but the C259S and C259N variants were very potently inhibited, the stoichiometry for parental E1 being about 1.6 mol of inhibitor/mol of E1 subunit. The C259S and C259N E1 variants required at least 25-fold greater inhibitor concentration to achieve the same level of inhibition. C259 is located in the putative thiamin diphosphate-binding motif of the enzyme [more exactly, it is adjacent to a ligand to the Mg(II) ion]. It is therefore concluded that thiamin 2-thiothiazolone diphosphate is not a transition-state analogue; rather, it is a potent inhibitor of the complex because of a specific interaction with the C259 residue.

Benzyloxycarbonylprolylprolinal, a transition-state analogue for prolyl oligopeptidase, forms a tetrahedral adduct with catalytic serine, not a reactive cysteine.

N-Benzyloxycarbonyl-l-prolyl-l-[1-13C]prolinal was synthesized starting with reduction of l-[1-13C]Pro to l-[1-13C]prolinol, followed by coupling with N-benzyloxycarbonyl-l-Pro to N-benzyloxycarbonyl-l-Pro-l-[1-13C]prolinol (Z-Pro-[1-13C]prolinol), and finally oxidation of the alcohol to the aldehyde with dimethyl sulphoxide. While the 13C NMR chemical shift of the aldehyde carbon is 202 p.p.m., that of the aldehyde hydrate is between 91.6 and 91.8 p.p.m., that of the dithiothreitol adduct is between 74.8 and 75.0 p. p.m., and that in the presence of the serine protease prolyl oligopeptidase is at 92.3 p.p.m.. The linewidth of the latter is 114 Hz, roughly consistent with the molecular mass of 80 kDa reported for the enzyme. Inverse detection experiments gave a 1H resonance at 5.29 p.p.m. with a linewidth of 80 Hz, also consistent with the expected chemical shift and linewidth for a hemiacetal bound to such a large enzyme, while the free hydrate gave resonances at 5.18 and 5. 25 p.p.m., with very much narrower linewidths. It is concluded that Z-Pro-prolinal, a putative transition-state analogue for prolyl oligopeptidase, forms a tetrahedral complex with the enzyme at its catalytic serine, rather than at a neighbouring cysteine that was found to be highly reactive according to chemical modification studies.

10] X-ray structures of retroviral proteases and their inhibitor-bound complexes

Publisher Summary This chapter focuses on the structure-based design strategies employed to inhibit the HIV protease. The design strategy takes two general forms: (1) determination of crystal structures of complexes between the protease and compounds with known inhibitory potency to define characteristics leading to efficient binding and (2) theoretical approaches using the known crystal structure of the protease to predict structures of inhibitory compounds followed by experimental verification. It is difficult to find patterns that lead obviously to high affinity if the peptide-based HIV protease inhibitors are considered. The general principle that increasing the hydrophobicity of the inhibitor leads to tighter binding seems to be established, but the question of the role of symmetry remains unresolved. Putative transition state analogs are effective inhibitors because they add at least one strong hydrogen bond but studies of these compounds have not established the mechanism of the enzyme nor have they confirmed the hypothesis that their tight binding results from their resemblance to the activated complex.

Observation of tightly bound boron-11 nuclear magnetic resonance signals on serine proteases. Direct solution evidence for tetrahedral geometry around the boron in the putative transition-state analogs

Observation of tightly bound boron-11 nuclear magnetic resonance signals on serine proteases. Direct solution evidence for tetrahedral geometry around the boron in the putative transition-state analogs

Mechanistically significant diastereoselection in the sulfoximine inhibition of carboxypeptidase A.

The diastereomers of L-alpha-[ [S-(2-phenylethyl)sulfonimidoyl]methyl]benzenepropanoic acid bind differentially to carboxypeptidase A. These putative transition state-analogue inhibitors show unique and interpretationally significant pH dependences for Ki, as well as for the visible absorption spectra of their E.I complexes in the case of the cobalt-substituted enzyme. From the geometry of the enzymically preferred isomer, it may be concluded that the mechanism of peptide scission by the enzyme entails addition of a nucleophile to the si face of the bound-substrate prochiral carboxamide linkage. New interpretational constraints on the mode of action of the enzyme are thereby imposed.

Mechanistic deductions from multiple kinetic and solvent deuterium isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: Escherichia coli tryptophan indole-lyase

Analysis of the pH dependence of the kinetic parameters and competitive inhibitor Ki values for tryptophan indole-lyase suggests two enzymic groups must be unprotonated in order to facilitate binding and catalysis of tryptophan. The V/K for tryptophan and the pKi for oxindolyl-L-alanine, a putative transition state analogue and competitive inhibitor, decrease below two pK values of 7.6 and 6.0, while the Ki for L-alanine, also a competitive inhibitor, is 3300-fold larger (20 mM) than that for oxindolyl-L-alanine and increases below a single pK of 7.6. A single pK of 7.6 is also observed in the V/K profile for the alternate substrate, S-methyl-L-cysteine. Therefore, the enzymic group with a pK of 7.6 is responsible for proton abstraction at the 2-position of tryptophan, while the enzymic group with a pK of 6.0 interacts with the indole portion of tryptophan and probably catalyzes formation of the indolenine tautomer of tryptophan (in concert with proton transfer to C-3 of indole from the group with pK 7.6) to facilitate carbon-carbon bond cleavage and elimination of indole. The pH variation of the primary deuterium isotope effects for proton abstraction at the 2-position of tryptophan (DV = 2.5 and D(V/Ktrp) = 2.8) are pH independent, while the Vmax for tryptophan or S-methyl-L-cysteine is the same and also pH independent. Thus, substrates bind only to the correctly protonated form of the enzyme. Further, tryptophan is not sticky, and the pK values observed in both V/K profiles are the correct ones.(ABSTRACT TRUNCATED AT 250 WORDS)

Ligand-mediated conformational changes in wheat-germ aspartate transcarbamoylase indicated by proteolytic susceptibility.

Ligand-mediated effects on the inactivation of pure wheat-germ aspartate transcarbamoylase by trypsin were examined. Inactivation was apparently first-order in all cases, and the effects of ligand concentration on the pseudo-first-order rate constant, k, were studied. Increase in k (labilization) was effected by carbamoyl phosphate, phosphate and the putative transition-state analogue, N-phosphonoacetyl-L-aspartate. Decrease in k (protection) was effected by the end-product inhibitor, UMP, and by the ligand pairs aspartate/phosphate and succinate/carbamoyl phosphate, but not by aspartate or succinate alone up to 10 mM. Except for protection by the latter ligand pairs, all other ligand-mediated effects were also observed on inactivation of the enzyme by Pronase and chymotrypsin. Ligand-mediated effects on the fragmentation of the polypeptide chain by trypsin were examined electrophoretically. Slight labilization of the chain was observed in the presence of carbamoyl phosphate, phosphate and N-phosphonoacetyl-L-aspartate. An extensive protection by UMP was observed, which apparently included all trypsin-sensitive peptide bonds. No significant effect by the ligand pair succinate/carbamoyl phosphate was noted. It is concluded from these observations that UMP triggers an extensive, probably co-operative, transition to a proteinase-resistant conformation, and that carbamoyl phosphate similarly triggers a transition to an alternative, proteinase-sensitive, conformation. These antagonistic conformational changes may account for the regulatory kinetic effects reported elsewhere [Yon (1984) Biochem. J. 221, 281-287]. The protective effect by the ligand pairs aspartate/phosphate and succinate/carbamoyl phosphate, which operates only against trypsin, is concluded to be due to local shielding of essential lysine or arginine residues in the aspartate-binding pocket of the active site, to which aspartate (or its analogue, succinate) can only bind as part of a ternary complex.

ChemInform Abstract: PURIFICATION OF RIBOFLAVIN SYNTHASE BY AFFINITY CHROMATOGRAPHY USING 7-OXOLUMAZINES

Riboflavin synthase (EC. 2.5.1.9) from baker's yeast has been purified over 1000 fold by affinity chromatography using the putative transition-state analogue 6-carboxyethyl-7-oxo-8-D-ribityllumazine as the ligand attached to Sepharose 4B.

Purification of riboflavin synthase by affinity chromatography using 7-oxolumazines

Riboflavin synthase (EC. 2.5.1.9) from baker's yeast has been purified over 1000 fold by affinity chromatography using the putative transition-state analogue 6-carboxyethyl-7-oxo-8-D-ribityllumazine as the ligand attached to Sepharose 4B.