Chemicals modification studies

Some chemical modification studies on the sea anemone toxins have unfortunately been less than rigorous in analyzing the reaction products. Consequently, results from many of these studies can only provide suggestions, rather than firm conclusions, regarding the importance of particular sidechains. Many such studies also have failed to determine if the secondary and tertiary structures of the toxin products were affected by chemical modification. 

Neurotoxins present in sea snake venoms are summarized. All sea snake venoms are extremely toxic, with low LD5Q values. Most sea snake neurotoxins consist of only 60-62 amino acid residues with 4 disulOde bonds, while some consist of 70 amino acids with 5 disulfide bonds. The origin of toxicity is due to the attachment of 2 neurotoxin molecules to 2 a subunits of an acetylcholine receptor that is composed of a2 6 subunits. The complete structure of several of the sea snake neurotoxins have been worked out. Through chemical modification studies the invariant tryptophan and tyrosine residues of post-synaptic neurotoxins were shown to be of a critical nature to the toxicity function of the molecule. Lysine and arginine are also believed to be important. Other marine vertebrate venoms are not well known. 

Chemical modification studies with fluorescein-5 -isothiocyanate support the proximity of Lys515 to the ATP binding site [98,113-117,212,339]. Fluorescein-5 -isothiocyanate stoichiometrically reacts with the Ca -ATPase in intact or solubilized sarcoplasmic reticulum at a mildly alkaline pH, causing inhibition of ATPase activity, ATP-dependent Ca transport, and the phosphorylation of the Ca " -ATPase by ATP the Ca uptake energized by acetylphosphate, carbamylphos-phate or j -nitrophenyl phosphate is only partially inhibited [113,114,212,339]. The reaction of -ATPase with FITC is competitively inhibited by ATP, AMPPNP, TNP-ATP, and less effectively by ADP or ITP the concentrations of the various nucleotides required for protection are consistent with their affinities for the ATP binding site of the Ca -ATPase [114,212,340]. 

Most of the antiemetic clinical trials in the last decade have involved metoclopramide (1) either as a single agent or in combination with other drugs. Similarly, most of the chemical modification studies have been designed to optimize antiemetic and/or gastroprokinetic properties of metoclopramide and to eliminate undesirable CNS side-effects which are the consequence of its dopamine D2 receptor blockade [1-3]. 

The fluorescence decay is multiexponential.(199 200) This is unequivocal evidence that the wyebutine base can be bound in at least two different conformations with different solvent shielding. Wells and Lakowicz(200) resolved two exponential components. They measured the normalized amplitudes and lifetimes for the wyebutine fluorescence at two different concentrations of added Mg2+ S° = 0.50, t, = 1.7 ns, = 0.50, and t2 = 5.9 ns with no added Mg2+ present, and S°i = 0.16, t, =0.6 ns, S2 = 0.84, and r2 = 6.0ns with 10 nM Mg2+. Since the 6ns component is the longest lifetime present, it must represent the conformation that shields the wyebutine to the greatest extent and is generally believed to involve a 3 stack of bases 34-38.w 199-2011 The fraction of the tRNA in this conformation increases when Mg2 + is added to the solution. This structure is also observed in crystal structures which include Mg2+.(202 204) In the other conformation(s), the wyebutine is more exposed to the solvent. A 5 stack, which does not include bases 37 and 38, is one possibility. The wyebutine base would be more exposed to the solvent and have a shorter fluorescence lifetime as a result. However, both NMR data(205 206) and chemical modification studies(207) are inconsistent with a 5 stack. For the moment, this matter is unresolved. 

Virtually all of what is known about the secondary and tertiary structure of tubulin has been gleaned from a limited number of spectroscopic and chemical modification studies. Failure to obtain crystals of suitable quality for X-ray diffraction studies likely results from both heterogeneity in the subunits and the propensity of tubulin to polymerize into many polymorphs (see Section III). George et al. (1981) reported that as many as 17 distinct protein peaks may be discerned after isoelectric focusing of purified tubulin. 

A pathway (Scheme I) (8.9) for the hydrolysis of oligoglycosides by lysozyme that differs from the previously accepted mechanism (Scheme II) (3,10-12) is described in this section. The alternative pathway, suggested by results of a 55-ps MD simulation of the lysozyme (GlcNAc)e complex (1), is consistent with the available experimental data and with stereoelectronic considerations. Experimental data have demonstrated that Glu 35 and Asp 52 are essential, as shown by recent site-directed mutagenesis results (13.) which corroborate chemical modification studies (3.14 and references cited therein), and that the reaction proceeds with retention of configuration at Ci Q and references cited therein). A fundamental feature of the alternative pathway is that an endocyclic bond is broken in the initial step, in contrast to the exocyclic bond cleavage in the accepted mechanism. 

The core-enzymes, prepared in our laboratory, and containing the active centers, were successfully crystallized (Dr. Jones, Uppsala, communicated) and tertiary structures will be described in the near future. Chemical modification studies on these enzymes are currently being undertaken in our laboratory identification of important catalytic residues and location of the active centers will lead to more functional information on these enzymes. Other cellulases such as some endoglucanases from Clostridium thermocel-lum (EG A, EG B, EG D) (10) and EngA and Exg from Cellulomonas fimi (19) also contain sequences of conserved, terminally located and sometimes reiterated, amino acids. Some of these sequences are preceded by proline-serine rich domains. Thus, a bistructural-bifunctional organization seems to be a rather common feature among cellulases, at least for EngA and Exg from C. fimi and the enzymes from Trichoderma reesei. 

The binding of one Zn ", presumably at the Zn site, organizes the active site, as shown with BESOD by H NMR spectroscopy and by chemical modification studies with diethylpyrocarbonate The ligands of the Zn rite are close together in the sequence (Scheme 2). 

The sulfur atom of methionine residues may be modified by formation of sulfonium salts or by oxidation to sulfoxides or the sulfone. The cyanosulfonium salt is not particularly useful for chemical modification studies because of the tendency for cyclization and chain cleavage (129). This fact, of course, makes it very useful in sequence work. Normally, the methionine residues of RNase can only be modified after denaturation of the protein, i.e., in acid pH, urea, detergents, etc. On treatment with iodoacetate or hydrogen peroxide, derivatives with more than one sulfonium or sulfoxide group did not form active enzymes on removal of the denaturing agent (130) [see, however, Jori et al. (131)]. There was an indication of some active monosubstituted derivatives (130, 132). 

The role of certain residues in the enzyme mechanism has been confirmed by chemical modification studies, notably for tyrosine. 14 Modification of tyrosyl residues (for example acetylation or nitration) leads to loss of peptidase activity and enhancement of esterase activity. The presence of the inhibitor -phenylpropionate protects two tyrosine residues from acetylation. Those are Tyr-248 and probably Tyr-198, which is also in the general area of the active site. The modified apoenzyme has lower affinity for dipeptides, as might be expected from the loss of hydrogen bonding between Tyr-248 and the peptide NH group. 

The chemical modification studies have thus not yet led to a much more conclusive picture of the active site than that outlined in Fig. 12, and the identification of amino acid side chains involved in catalysis or substrate binding may have to await the completion of the crystal structure determination. The reporter properties of the Co(II) enzyme clearly show, however, that an open coordination position is of decisive functional importance, that the metal ion is intimately associated with the basic group participating in the reaction, and that the metal ion is probably also involved in the binding of one of the substrates, HCO3. 

Affinity labeling and chemical modification studies were carried out with purified Fi. The /3Thr-287, /3Ile-290 and /3Tyr-297 residues ( coli numbering) of beef heart Ft were... 

On the basis of chemical modification studies, Tyr 198 of carboxypeptidase A was proposed to act as a proton donor (i.e., a general acid) in the mechanism of catalysis. However, when Tyr 198 was replaced with Phe by means of site-directed mutagenesis, the modified enzyme retained substantial enzymatic activity, indicating that the tyrosyl hydroxyl may not have a specific role in catalysis. 

Chemical modification studies on ADPGlc PPase have involved the use of the following affinity labels ... 

The amino acid sequence of the protein, which can be inferred from the published cDNA sequence (Edman et ai, 1985), reveals that each PDI polyjjeptide contains two domains that are closely homologous to thioredoxin. This enzyme has been characterized in detail (Holmgren, 1985) and it has been shown that the cysteine residues in the sequence WCGPCK (residues 31-36) act as the reactive dithiol. Chemical modification studies have shown that the enzyme, like PDI, is inactivated by alkylation at neutral pH, and that only Cys-32 is alkylated (Kallis and... 

A combination of steady-state and presteady sate kinetic and spectral and chemical modification studies led to a mechanistic scheme in which inhibition occurs through the binding of the zinc-monohydroxide species to the active EH species of the enzyme (Scheme 2) The pH independent constant for the inhibition by zinc is 0.71 J,M. The derived p/fa of 6 for the inhibition studies agrees with the corresponding value obtained in peptide hydrolysis experiments for the group, EH2, whose ionization leads to formation of the catalytically active form of the enzyme. 

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