George L. Kenyon

[40] Novel sulfhydryl reagents

Publisher Summary This chapter focuses on sulfhydryl reagents that have been categorized as blocking and labeling groups, reporter groups, cross-linking groups, and affinity labeling groups. Most of these sulfhydryl reagents deliver groups that fall in the category of “blocking and labeling groups,” which may be either reversible or irreversible. These groups are designed to be structurally and chemically relatively innocuous so that when covalently bound, they act merely to block the activity of or to titrate or label (sometimes isotopically) any number of sulfhydryl groups. A second class of sulfhydryl reagents delivers cross-linking groups. They possess one functionality that reacts initially and selectively with sulfhydryls and a second functionality that reacts (often under altered conditions) with another nearby group, which may or may not be another sulfhydryl. For the other two major categories, reporter groups and affinity labeling groups, there is a scarcity of older reagents that may be similarly classified, which react satisfactorily, and are of any general practical utility.

A functionally diverse enzyme superfamily that abstracts the alpha protons of carboxylic acids

Mandelate racemase and muconate lactonizing enzyme are structurally homologous but catalyze different reactions, each initiated by proton abstraction from carbon. The structural similarity to mandelate racemase of a previously unidentified gene product was used to deduce its function as a galactonate dehydratase. In this enzyme superfamily that has evolved to catalyze proton abstraction from carbon, three variations of homologous active site architectures are now represented: lysine and histidine bases in the active site of mandelate racemase, only a lysine base in the active site of muconate lactonizing enzyme, and only a histidine base in the active site of galactonate dehydratase. This discovery supports the hypothesis that new enzymatic activities evolve by recruitment of a protein catalyzing the same type of chemical reaction.

On the origin of enzymatic species

The diversity of enzyme catalytic function is remarkable, particularly when one considers that ancestral life forms must have started with a much smaller ensemble of proteins. In this article, we discuss the evolution of the mandelate pathway in pseudomonads as an example of how catalytic diversity may have evolved. We suggest that existing enzymes that catalyse the chemistry needed to accomplish a transformation were recruited, followed by the evolution of specific binding.

Novel alkyl alkanethiolsulfonate sulfhydryl reagents. Modification of derivatives ofl-cysteine

A simple, general scheme for the synthesis of sulfhydryl-specific alkyl alkanethiolsulfonate (RSSO2R′) reagents where R′ is methyl, has been developed. Two new reagents, methyl aminoethanethiolsulfonate (2) and methyl benzylthiolsulfonate (3) were synthesized. These were used to modify stoichiometrically and selectively under mild conditions the sulfhydryl groups ofN-acetyl-l-cysteine ethyl ester (4),N-acetyl-l-cysteinep-nitroanilide (7), glutathione, and the A chain of bovine insulin. The corresponding β-S-(β-aminoethanethiol) and β-S-(benzylthiol) derivatives ofl-cysteine and of the peptides were afforded. The characteristics and significance of these reactions and products are discussed.

Chalcone, acyl hydrazide, and related amides kill cultured Trypanosoma brucei brucei.

BackgroundProtozoan parasites of the genus Trypanosoma cause disease in a wide range of mammalian hosts. Trypanosoma brucei brucei, transmitted by tsetse fly to cattle, causes a disease (Nagana) of great economic importance in parts of Africa. T. b. brucei also serves as a model for related Trypanosoma species, which cause human sleeping sickness.Materials and MethodsChalcone and acyl hydrazide derivatives are known to retard the growth of Plasmodium falciparum in vitro and inhibit the malarial cysteine proteinase, falcipain. We tested the effects of these compounds on the growth of bloodstream forms of T. b. brucei in cell culture and in a murine trypanosomiasis model, and investigated their ability to inhibit trypanopain-Tb, the major cysteine proteinase of T. b. brucei.ResultsSeveral related chalcones, acyl hydrazides, and amides killed cultured bloodstream forms of T. b. brucei, with the most effective compound reducing parasite numbers by 50% relative to control populations at a concentration of 240 nM. The most effective inhibitors protected mice from an otherwise lethal T. b. brucei infection in an in vivo model of acute parasite infection. Many of the compounds also inhibited trypanopain-Tb, with the most effective inhibitor having a Ki value of 27 nM. Ki values for trypanopain-Tb inhibition were up to 50- to 100-fold lower than for inhibition of mammalian cathepsin L, suggesting the possibility of selective inhibition of the parasite enzyme.ConclusionsChalcones, acyl hydrazides, and amides show promise as antitrypanosomal chemotherapeutic agents, with trypanopain-Tb possibly being one of their in vivo targets.

Anti-malarial drug development using models of enzyme structure

BACKGROUND The trophozoite stage of the malaria parasite infects red blood cells. During this phase of their life-cycle, the parasites use hemoglobin as their principal source of amino acids, using a cysteine protease to degrade it. We have previously reported a three-dimensional model of this cysteine protease, based on the structures of homologous proteases, and the use of the program DOCK to identify a ligand for the malaria protease. RESULTS Here we describe the design of improved ligands starting from this lead. Ligand design was based on the predicted configuration of the lead compound docked to the model three-dimensional structure of the protease. The lead compound has an IC50 of 6 microM, and our design/synthesis strategy has resulted in increasingly potent derivatives that block the ability of the parasites to infect and/or mature in red blood cells. The two best derivatives to date have IC50(s) of 450 nM and 150 nM. CONCLUSIONS A new class of anti-malarial chemotherapeutics has resulted from a computational search that was based on a model of the target protease. Despite the lack of a detailed experimental structure of the target enzyme or the enzyme-inhibitor complex, we have been able to identify compounds with increased potency. These compounds approach the activity of chloroquine (IC50 = 20 nM), but have a distinct mechanism of action. This series of compounds could thus lead to new therapies for chloroquine-resistant malaria.

Reversible modification of the sulfhydryl groups of Escherichia coli succinic thiokinase with methanethiolating reagents, 5,5′-dithio-bis(2-nitrobenzoic acid), p-hydroxymercuribenzoate, and ethylmercurithiosalicylate

Abstract Inhibition of Escherichia coli succinic thiokinase by either methyl methanethiolsulfonate (MeS-SO 2 Me) or by methoxycarbonylmethyl disulfide was reversed completely by addition of tributylphosphine, but not by incubation with dithiothreitol. At a point where about 4 moles of MeS- were incorporated per mole of enzyme ( M r 140,000), no loss of antigenicity (as measured by microcomplement fixation) was observed, but at least 80% of thiokinase activity was lost. No significant conformational change was indicated for this methanethiolated enzyme in the ultracentrifuge. In contrast, losses of both thiokinase activity and antigenicity were brought about by incubation with 5,5′-dithiobis 2-nitrobenzoic acid) (Nbs 2 ), ethylmercurithiosalicylate, and p -hydroxymercuribenzoate. These losses were restored totally by incubation of modified enzyme with dithiothreitol but not with tributylphosphine. Preincubation of succinic thiokinases with MeSSO 2 Me and with Nbs 2 protected the enzyme partially, and to the same degree, against the inhibiting effects of N -ethylmaleimide. These data suggest that, while both MeSSO 2 Me and Nbs 2 may attack identical thiol groups on the enzyme, each may also react specifically with other thiol groups. Inhibition by MeS-SO 2 Me and by Nbs 2 both appear not to be accompanied by dissociation into subunits and, in the later case, significant aggregation appears to be involved.

Comparisons of creatine kinase primary structures

Comparisons of nine creatine kinase sequences show that 67% of the protein sequence is identical among rabbit, rat, mouse, and chicken muscle, rabbit, rat, and chicken brain, and electric organ sequences from two species of electric ray(Torpedo). The extensive homology precludes a facile prediction of active-site residues based on sequence conservation. The sequences are more similar within isozyme types than are the different isozymes from any one species. There are 35 positions in the muscle and brain sequence pairs for three species which differentiate the two forms. TheTorpedo sequences do not fall completely into either of these patterns. Except for homology with partial sequences of other ATP-guanidino phosphotransferases, no significant homology with other protein or nucleic acid sequences in available databases was found. Preliminary secondary structural predictions suggest that the C-terminal half of the protein is likely an α/β-type protein. Placement in the sequence of two peptides found in previous cross-linking studies reveals two stretches of primary structure that are presumably close in space to the reactive Cys-283 and hence close to the active site.

Saturation mutagenesis of putative catalytic residues of benzoylformate decarboxylase provides a challenge to the accepted mechanism

Benzoylformate decarboxylase from Pseudomonas putida (PpBFDC) is a thiamin diphosphate-dependent enzyme that carries out the nonoxidative decarboxylation of aromatic 2-keto acids. The x-ray structure of PpBFDC suggested that Ser-26, His-70, and His-281 would play important roles in its catalytic mechanism, and the S26A, H70A, and H281A variants all exhibited greatly impaired catalytic activity. Based on stopped-flow studies with the alanine mutants, it was proposed that the histidine residues acted as acid-base catalysts, whereas Ser-26 was involved in substrate binding and played a significant, albeit less well defined, role in catalysis. While developing a saturation mutagenesis protocol to examine residues involved in PpBFDC substrate specificity, we tested the procedure on His-281. To our surprise, we found that His-281, which is thought to be necessary for protonation of the carbanion/enamine intermediate, could be replaced by phenyl alanine with only a 5-fold decrease in kcat. Even more surprising were our subsequent observations (i) that His-70 could be replaced by threonine or leucine with approximately a 30-fold decrease in kcat/Km compared with a 4,000-fold decrease for the H70A variant and (ii) that Ser-26, which forms hydrogen bonds with the substrate carboxylate, could be replaced by threonine, leucine, or methionine without significant loss of activity. These results call into question the assigned roles for Ser-26, His-70, and His-281. Further, they demonstrate the danger in assigning catalytic function based solely on results with alanine mutants and show that saturation mutagenesis is a valuable tool in assessing the role and relative importance of putative catalytic residues.

Probing the Active Center of Benzaldehyde Lyase with Substitutions and the Pseudosubstrate Analogue Benzoylphosphonic Acid Methyl Ester

Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of ( R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg (2+) as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these types of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analogue of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 A (Protein Data Bank entry 3D7K ) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase.