Functional groups on the cleavage site pyrimidine nucleotide are required for stabilization of the hammerhead transition state.

The role of individual functional groups on cytidine 17 in the hammerhead ribozyme was assessed by introducing modified pyrimidines into two kinetically well-characterized hammerheads. As long as the pyrimide ring size was maintained, the modifications had no effect on substrate binding, suggesting that the C17-C3 hydrogen bond observed in the X-ray structure is energetically neutral. However, modification of the exocyclic amino group and the carbonyl of C17 reduced the cleavage rate significantly, indicating that these groups are important in stabilizing the transition-state structure. C17 modifications did not affect the ratio of the forward and reverse reaction rates. Thus, unlike that believed previously, C17 is another one of many hammerhead residues critical in maintaining its active structure.     

Structure of the sodium borohydride-reduced N-(cyclopropyl)glycine adduct of the flavoenzyme monomeric sarcosine oxidase

Monomeric sarcosine oxidase (MSOX) is a flavoprotein that contains covalently bound FAD [8a-(S-cysteinyl)FAD] and catalyzes the oxidation of sarcosine (N-methylglycine) and other secondary amino acids, such as l-proline. Our previous studies showed that N-(cyclopropyl)glycine (CPG) acts as a mechanism-based inactivator of MSOX [Zhao, G., et al. (2000) Biochemistry 39, 14341-14347]. The reaction results in the formation of a modified reduced flavin that can be further reduced and stabilized by treatment with sodium borohydride. The borohydride-reduced CPG-modified enzyme exhibits a mass increase of 63 +/- 2 Da as compared with native MSOX. The crystal structure of the modified enzyme, solved at 1.85 A resolution, shows that FAD is the only site of modification. The modified FAD contains a fused five-membered ring, linking the C(4a) and N(5) atoms of the flavin ring, with an additional oxygen atom bound to the carbon atom attached to N(5) and a tetrahedral carbon atom at flavin C(4) with a hydroxyl group attached to C(4). On the basis of the crystal structure of the borohydride-stabilized adduct, we conclude that the labile CPG-modified flavin is a 4a,5-dihydroflavin derivative with a substituent derived from the cleavage of the cyclopropyl ring in CPG. The results are consistent with CPG-mediated inactivation in a reaction initiated by single electron transfer from the amine function in CPG to FAD in MSOX, followed by collapse of the radical pair to yield a covalently modified 4a,5-dihydroflavin.     

Structure of a stable form of sulfheme

A stable green heme was extracted from ferric cyanosulfmyoglobin after it had undergone an internal conversion reaction. After iron removal and conversion to the methyl ester, the resulting green porphyrin was purified by high-pressure liquid chromatography. Visible, 1H NMR, and mass spectrometric studies provided evidence to identify the substituents of the porphyrin. Nuclear Overhauser enhancements enabled an assignment of the single modified pyrrole. Substituent positions 1, 2, 5, 6, 7, and 8 have the original protoporphyrin IX substituents. At ring B, the 4-vinyl group has cyclized with a single sulfur atom to form a fifth ring with a 2,5-dihydrothiophene type of structure.     

Affinity labeling of the allosteric site of fructose 1,6-bisphosphatase with an AMP analog

D-Fructose 1,6-bisphosphatase [EC 3.1.3.11, FBPase] is one of the key enzymes in glyconeogenesis and its activity is controlled by various effectors such as substrate, AMP and ATP. To analyze this complex regulation system, we tried an affinity labeling of FBPase with an AMP derivative, since AMP is a potent allosteric inhibitor of this enzyme. The results obtained are as follows. 1. To determine the functional groups which are essential for AMP as an inhibitor, inhibitory activities of some AMP derivatives were examined. These derivatives modified at the purine ring or phosphate group lost the activity while one modified at the ribose ring retained the ability to inhibit FBPase. This shows that an affinity labeling reagent should be an AMP derivative in which the ribose ring is modified. 2. 2',3'-Dialdehyde AMP (dial-AMP) was prepared by periodate oxidation of AMP and was reacted with FBPase. Under appropriate conditions, 1 mol of the reagent was incorporated per mol of enzyme subunit with a concomitant loss of enzyme activity. The reaction was prevented by the presence of AMP but not of ATP. The heat-stability, the kinetic parameters and the UV-absorption spectrum of the modified enzyme were all the same as those of native FBPase in the presence of AMP. Thus it was concluded that the allosteric AMP site in FBPase was modified specifically.     

Ab initio molecular dynamics simulations of beta-D-glucose and beta-D-xylose degradation mechanisms in acidic aqueous solution

Ab initio molecular dynamics simulations were employed to investigate, with explicit solvent water molecules, beta-D-glucose and beta-D-xylose degradation mechanisms in acidic media. The rate-limiting step in sugar degradation was found to be protonation of the hydroxyl groups on the sugar ring. We found that the structure of water molecules plays a significant role in the acidic sugar degradation pathways. Firstly, a water molecule competes with the hydroxyl group on the sugar ring for protons. Secondly, water forms hydrogen bonds with the hydroxyl groups on the sugar rings, thus weakening the C-C and C-O bonds (each to a different degree). Note that the reaction pathways could be altered due to the change of relative stability of the C-C and C-O bonds. Thirdly, water molecules that are hydrogen-bonded to sugar hydroxyls could easily extract a proton from the reaction intermediate, terminating the reaction. Indeed, the sugar degradation pathway is complex due to multiple protonation probabilities and the surrounding water structure. Our experimental data support multiple sugar acidic degradation pathways.     

An activation mechanism for ATP cleavage in muscle

Evidence for a proposed activation mechanism is summarized. The low rate of ATP cleavage in the resting state of muscle is considered to result from the formation of a stable ring structure involving the two essential sulfhydryl groups on each myosin head and MgATP. Activation is thought to occur by interaction of actin in the vicinity of one of the essential sulfhydryl groups, Thus opening the stable ring leading to rapid dissociation of split products. This idea is consistent with the kinetic scheme of ATP cleavage developed recently by other workers and allows a prediction of the shift in population of intermediate states with changes in solvent conditions. It is also supported by our recent studies on the spatial geometry of the ring. The possibility that other nucleophilic groups may replace the sulfhydryl groups in other contractile systems is considered. The relevance of the ring structure to the tension generating event is discussed on the basis of recent measurements of the rate of contraction of modified (SH₁-blocked) actomyosin threads. Results indicate that the ability to form the ring structure is an essential requirement of the contractile process in these systems, and, moreover, that single, modified heads of myosin can act independently to produce the same rate of contraction as native myosin. This latter finding suggests that the myosin duplex exhibits some type of negative cooperativity in the contractile process.     

Approaches to the total synthesis of biologically active natural products: studies directed towards bryostatins

Progress on a total synthesis of the marine natural products, the bryostatins, is reviewed. Following studies aimed at the synthesis of the 1,16- and 17,27-fragments, procedures for the assembly of the macrocyclic ring of the bryostatins were investigated. Although ring-closing metathesis was not found to be useful for the synthesis of bryostatins with geminal dimethyl groups at C18, the modified Julia reaction was found to be useful for the stereoselective formation of the 16,17-double-bond and led to a synthesis of an advanced macrocyclic intermediate. Several novel synthetic procedures feature in this work.     

Site-directed mutagenesis and X-ray crystallography of the PQQ-containing quinoprotein methanol dehydrogenase and its electron acceptor, cytochrome c(L)

Two proteins specifically involved in methanol oxidation in the methylotrophic bacterium Methylobacterium extorquens have been modified by site-directed mutagenesis. Mutation of the proposed active site base (Asp303) to glutamate in methanol dehydrogenase (MDH) gave an active enzyme (D303E-MDH) with a greatly reduced affinity for substrate and with a lower activation energy. Results of kinetic and deuterium isotope studies showed that the essential mechanism in the mutant protein was unchanged, and that the step requiring activation by ammonia remained rate limiting. No spectrally detectable intermediates could be observed during the reaction. The X-ray structure, determined to 3 A resolution, of D303E-MDH showed that the position and coordination geometry of the Ca2+ ion in the active site was altered; the larger Glu303 side chain was coordinated to the Ca2+ ion and also hydrogen bonded to the O5 atom of pyrroloquinoline quinone (PQQ). The properties and structure of the D303E-MDH are consistent with the previous proposal that the reaction in MDH is initiated by proton abstraction involving Asp303, and that the mechanism involves a direct hydride transfer reaction. Mutation of the two adjacent cysteine residues that make up the novel disulfide ring in the active site of MDH led to an inactive enzyme, confirming the essential role of this remarkable ring structure. Mutations of cytochrome c(L), which is the electron acceptor from MDH was used to identify Met109 as the sixth ligand to the heme.     

An investigation of the carboxyl group function in the active center of transketolase

Transketolase from baker's yeast is rapidly inactivated in the presence of 1-ethyl-3 (3'-dimethylaminopropyl)-carbodiimide. pKa of the modified carboxyl groups is approximately 6.5. An investigation of the initial steps of enzymatic catalysis monitored by a changes in the circular dichroism spectra and in an oxidation reaction with ferricyanide made it possible to conclude that the modification interferes with the donor substrate attachment to the enzyme. Evidence obtained was suggesting that the carboxyl group of the active center facilitates dissociation of a proton from the carbon atom in the second position of the thiamine pyrophosphate thiazolium ring.     

Spin-label study of the mobility of enzyme-bound lipoic acid in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The lipoic acid residues covalently bound to the transacetylase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli were selectively modified by reaction with 4-maleimido-2,2,6,6-tetramethylpiperidino-oxyl. The electron-spin-resonance spectrum of the spin-labelled enzyme indicates that the bound nitroxide groups have high mobilities relative to the protein molecule. This physicochemical evidence is consistent with the view that the dithiolane ring of a lipoyl residue is capable of rapid migration between the active sites of the component enzymes in the catalytic mechanism.     

Variability in the carbazole assay for N-desulfonated/N-acylated heparin derivatives

The carbazole assay is commonly employed to quantify heparin and other uronic acid-containing polysaccharides. Heparin-derived standard curves are often employed to quantify solutions of various natural and unnatural heparin structures that have different levels of sulfate substitution, different levels of N-sulfo and N-acetyl groups, and other structural changes as a consequence of reducing molecular weight. Recent studies in our laboratory have focused on chemically modified heparin derivatives comprised of structurally diverse N-acyl moieties substituted into heparin in place of N-sulfo groups. We report here that although differing degrees of 2-N-sulfo-, 2-N-acetyl- or 2-amino-d-glucosamine residues within heparin do not affect signal intensity in the carbazole assay, replacing N-sulfo groups in heparin with structurally diverse N-acyl moieties affords products that display significant variation in the assay. The structure of different N-acyl groups, and to a lesser extent the degree of N-acylation by individual N-acyl groups, is shown to variably alter signal intensity in the carbazole assay even though content and structure of uronic acid residues is unaltered.     

Stereoselective synthesis of conformationally rigid apio carbanucleosides as potential antiviral agents

Apio north-methanocarbocyclic nucleosides 1-3 with bicyclo[3. 1.0]hexane template were first synthesized. Introduction of hydroxymethyl substituent was efficiently and stereoselectively accomplished by aldol and retro-aldol reaction and fixed conformation was achieved from a modified Simmons-Smith cyclopropanation on a cyclopentane ring.     

Molecular modeling reveals the possible importance of a carbonyl oxygen binding pocket for the catalytic mechanism of p-hydroxybenzoate hydroxylase

p-Hydroxybenzoate hydroxylase catalyzes the hydroxylation of an aromatic substrate and uses flavin as a cofactor. The reaction probably occurs via a flavin 4a-hydroperoxide intermediate. In this study the crystal structure of 4a,5-epoxyethano-3-methyl-4a,5-dihydrolumiflavin, an analogue of the flavin 4a-hydroperoxide intermediate, was fitted to the active site in the crystal structure of the p-hydroxybenzoate hydroxylase-3,4-dihydroxybenzoate complex. This model of an important catalytic intermediate fitted very well in the active site of p-hydroxybenzoate hydroxylase. The most striking result was that whereas with the normal flavin, the 0-4 of the flavin ring makes only poor hydrogen bonds with the protein, with the flavin 4a-hydroperoxide analogue, the same 0-4 makes strong hydrogen bonds with the NH groups of Gly-46 and Val-47. These two NH groups form a carbonyl oxygen binding pocket which has a geometry almost identical to the oxyanion hole found in several proteases. The possible consequences of this model for the reaction mechanism of p-hydroxybenzoate hydroxylase are discussed.     

Protein dynamics and electrostatics in the function of p-hydroxybenzoate hydroxylase

para-Hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor, FAD, by NADPH in response to binding p-hydroxybenzoate to the enzyme, then oxidation of reduced FAD by oxygen to form a hydroperoxide, which oxygenates p-hydroxybenzoate to form 3,4-dihydroxybenzoate. These diverse reactions all occur within a single polypeptide and are achieved through conformational rearrangements of the isoalloxazine ring and protein residues within the protein structure. In this review, we examine the complex dynamic behavior of the protein that enables regulated fast and specific catalysis to occur. Original research papers (principally from the past 15 years) provide the information that is used to develop a comprehensive overview of the catalytic process. Much of this information has come from detailed analysis of many specific mutants of the enzyme using rapid reaction technology, biophysical measurements, and high-resolution structures obtained by X-ray crystallography. We describe how three conformations of the enzyme provide a foundation for the catalytic cycle. One conformation has a closed active site for the conduct of the oxygen reactions, which must occur in the absence of solvent. The second conformation has a partly open active site for exchange of substrate and product, and the third conformation has a closed protein structure with the isoalloxazine ring rotated out to the surface for reaction with NADPH, which binds in a surface cleft. A fundamental feature of the enzyme is a H-bond network that connects the phenolic group of the substrate in the buried active site to the surface of the protein. This network serves to protonate and deprotonate the substrate and product in the active site to promote catalysis and regulate the coordination of conformational states for efficient catalysis.     

Mechanical unfolding of covalently linked GroES: Evidence of structural subunit intermediates

It is difficult to determine the structural stability of the individual subunits or protomers of many proteins in the cell that exist in an oligomeric or complexed state. In this study, we used single‐molecule force spectroscopy on seven subunits of covalently linked cochaperonin GroES (ESC7) to evaluate the structural stability of the subunit. A modified form of ESC7 was immobilized on a mica surface. The force‐extension profile obtained from the mechanical unfolding of this ESC7 showed a distinctive sawtooth pattern that is typical for multimodular proteins. When analyzed according to the worm‐like chain model, the contour lengths calculated from the peaks in the profile suggested that linked‐GroES subunits unfold in distinct steps after the oligomeric ring structure of ESC7 is disrupted. The evidence that structured subunits of ESC7 withstand external force to some extent even after the perturbation of the oligomeric ring structure suggests that a stable monomeric intermediate is an important component of the equilibrium unfolding reaction of GroES.     

Unexpected formation of complex bridged tetrazoles via intramolecular 1,3-dipolar cycloaddition of 1,2-O-cyanoalkylidene derivatives of 3-azido-3-deoxy-D-allose

An unexpected and interesting intramolecular side reaction occurred during the attempted synthesis of glycosyl cyanides upon treatment of 1-O-acetyl-3-azido-3-deoxyallose derivatives with TMSCN and different Lewis acids. Exo-1,2-O-cyanoalkylidene derivatives formed by neighboring group participation and attack of cyanide underwent, after Lewis-acid mediated isomerization to the endo-isomer, intramolecular azide-cyanide cycloaddition leading to the formation of tetrazoles embedded in bridged tetracyclic ring systems. The efficiency of cycloaddition is dependent on the ring structure of the sugar (pyranose or furanose). Of the studied molecules, 3-azido-1,2-O-cyanoethylidene-3-deoxy-allopyranose provides the most suitable scaffold for intramolecular [2+3] cycloaddition under exceptionally mild conditions. Our results highlight the capability of carbohydrates to act as scaffolds for the precise positioning of functional groups productive for a specific chemical reaction.     

Synthesis and evaluation of the performance of a small molecule library based on diverse tropane-related scaffolds

A unified synthetic approach was developed that enabled the synthesis of diverse tropane-related scaffolds. The key intermediates that were exploited were cycloadducts formed by reaction between 3-hydroxy-pyridinium salts and vinyl sulfones or sulfonamides. The diverse tropane-related scaffolds were formed by addition of substituents to, cyclisation reactions of, and fusion of additional ring(s) to the key bicyclic intermediates. A set of 53 screening compounds was designed, synthesised and evaluated in order to determine the biological relevance of the scaffolds accessible using the synthetic approach. Two inhibitors of Hedgehog signalling, and four compounds with weak activity against the parasite P. falciparum, were discovered. Three of the active compounds may be considered to be indotropane or pyrrotropane pseudo natural products in which a tropane is fused with a fragment from another natural product class. It was concluded that the unified synthetic approach had yielded diverse scaffolds suitable for the design of performance-diverse screening libraries.     

Inhibition of LDL oxidation by flavonoids in relation to their structure and calculated enthalpy

Twenty flavonoid compounds of five different subclasses were selected, and the relationship of their structure to the inhibition of low-density lipoprotein (LDL) oxidation in vitro was investigated. The most effective inhibitors, by either copper ion or 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH) induction, were flavonols and/or flavonoids with two adjacent hydroxyl groups at ring B. In the presence of the later catechol group, the contribution of the double bond and the carbonyl group at ring C was negligible. Isoflavonoids were more effective inhibitors than other flavonoid subclasses with similar structure. Substituting ring B with hydroxyl group(s) at 2' position resulted in a significantly higher inhibitory effect than by substituting ring A or ring B at other positions. The type of LDL inducer had no effect in flavonoids with catechol structure. Calculated heat of formation data (deltadeltaH(f)) revealed that the donation of a hydrogen atom from position 3 was the most likely result, followed by that of a hydroxyl from ring B. Position 3 was favored only in the presence of conjugated double bonds between ring A to ring B. This study makes it possible to assign the contribution of different functional groups among the flavonoid subclasses to in vitro inhibition of LDL oxidation.     

Rational design and synthesis of highly potent anti-acetylcholinesterase activity huperzine A derivatives

By targeting multi-active sites of acetylcholinesterase (AChE), a series of huperzine A (Hup A) derivatives with various aromatic ring groups were designed and synthesized by Schiff reaction. They were evaluated as AChE and butyrylcholinesterase (BChE) inhibitors. Results showed very significant specificity that the group of imine derivatives could inhibit TcAChE and hAChE, but no inhibitory effect on hBChE was detected. The experiment was explained by a docking study. In the docking model, we confirmed that aromatic ring of Hup A derivatives played the π–π stacking against aminophenol residues of AChE, and the structure–activity relationship (SAR) was discussed.     

Structure-Activity Relationships of Vasopressin Analogues on Release of Factor Viii in Dogs

Abstract

In order to study structure-activity relationships as to Factor VIII release conscious dogs were injected with analogues of vasopressin. The peptides used were chemically modified either in the hexapeptide ring structure of the vasopressin molecule or in the C-terminal tripeptide or in both. The results showed that an intact C-terminal appears to be of importance for retaining Factor VIII releasing activity of the analogues, whereas at least some modifications of the ring structure are tolerated without loss of activity. Decreased activity was also observed when the disulphide bridge was substituted with a monocarba bond.