The role of radicals in enzymatic processes

Research on the mechanism of action of coenzyme B12, adenosylcobalamin, as a graduate student introduced the author to the field of organic free radicals in enzymology. Twenty years later, related work on S-adenosylmethionine (SAM) as a "poor man's coenzyme B12" was initiated in a detailed analysis of the mechanism of action of lysine 2,3-aminomutase (LAM). The interconversion of L-lysine and L-beta-lysine is catalyzed by LAM, which requires SAM, pyridoxal-5'-phosphate (PLP), and a [4Fe-4S] cluster as coenzymes. The mechanism of this reaction has been delineated as a radical isomerization, in which radical formation is initiated by the [4Fe-4S]-dependent cleavage of the SAM into methionine and the 5'-deoxyadenosyl radical. The mechanism of this process is discussed, together with the role of this radical in hydrogen abstraction from lysine to initiate the substrate radical isomerization. The chemistry underlying the functions of SAM, PLP, and [4Fe-4S] in the action of LAM is novel in all respects, except for the formation of a lysine-PLP aldimine at the active site. Of the four free radicals in the mechanism, three have been characterized by EPR spectroscopy. In the suicide inactivation of adenosylcobalamin-dependent dioldehydrase (DDH) by glycolaldehyde, the formation of cob(II)alamin and 5'-deoxyadenosine is accompanied by the conversion of glycolaldehyde to cis-ethanesemidione radical at the active site. The cis-ethanesemidione radical has been characterized by EPR spectroscopy. Its exceptional stability at the active site is the basis for the inactivation of DDH by glycolaldehyde.     

Form fluctuations of carbosilane dendrimers in dilute solution: a study using neutron spin echo spectroscopy

A series of carbosilane dendrimers with perfluorinated end groups has been prepared. The structure of these molecules in dilute solution is studied using small angle neutron scattering. For generations g<3 we find a non-spherical shape of the particles and a tendency for aggregation. This result is supported by the analysis of the diffusion coefficient obtained from photon correlation spectroscopy. The overall shape of the molecules is that of a core-shell particle. The generation 4 molecule is obtained as a compact sphere. Neutron spin echo spectroscopy reveals a relaxation time which is attributed to the form fluctuations of this particle.     

Multiple isotope effect study of the acid-catalyzed hydrolysis of methyl formate

Multiple isotope effects have been measured for the acid-catalyzed hydrolysis of methyl formate in 0.5 M HCl at 20 degrees C. The isotope effects in the present investigation include the carbonyl carbon (13k = 1.028 +/- 0.001), the carbonyl oxygen (18k = 0.9945 +/- 0.0009), the nucleophile oxygen (18k = 0.995 +/- 0.001), and the formyl hydrogen ((D)k = 0.81 +/- 0.02). Determination of the carbonyl carbon, carbonyl oxygen, and formyl hydrogen isotope effects was performed via isotopic analysis of residual substrate. However, determination of the oxygen nucleophile isotope effect required analysis of the oxygen atoms of the product (formic acid), which exchange with the solvent (water) under acid conditions. This necessitated measurement of the rate of exchange of these oxygen atoms under the conditions for hydrolysis (k(ex) = 0.0723 min(-1)) and correction of the raw isotope ratios measured during the nucleophile-O isotope effect experiment. These results, along with the previously reported isotope effect for the leaving oxygen (18k = 1.0009) and the ratio of the rate of hydrolysis to that of exchange of the carbonyl oxygen with water (k(h)/k(ex) = 11.3), give a detailed picture of the transition-state structure for the reaction.     

Elucidating the mode of action of a corrosion inhibitor for iron

Two polymetallic iron(III) complexes 1 and 2 have been synthesised from the known corrosion inhibitor 3-(4-methylbenzoyl)-propionic acid HL1 and their crystal structures determined. Coordination geometries extracted from these structures have been used as the basis for molecular modelling onto idealised iron(III) oxide surfaces as an aid to understanding the efficacy of inhibitors of the 4-keto acid type. The proposed mode of action involves 1,3-bridging didentate coordination of the carboxylate function of L1 to two FeIII ions, hydrogen-bond formation between the 4-keto group of L1 and a bridging surface hydroxy group, as well as close packing of the aromatic end groups, which should generate a hydrophobic barrier on the surface. Adsorption isotherm experiments have been used to compare the strengths of binding of related carboxylic acids onto iron(III) oxide surfaces and indicate that the presence of the 4-keto function leads to the formation of significantly more stable surface complexes.     

Gold catalysis contra platinum catalysis in hydroarylation contra phenol synthesis

Three silylated γ-alkynylfurans were prepared and subjected to both gold and platinum catalysts. The TMS- and the TBDMS-substituted furans reacted. With AuCl₃ and the binuclear [(Ph₃PAu)₂Cl][BF₄] catalyst a hydroarylation of the alkyne was observed. Na[AuCl₄] gave phenols as the product, but these were formed only after in situ desilylation of the starting material by the gold catalyst and thus the wrong isomer dominated. Only with PtCl₂(MeCN)₂ phenols with a silyl group were formed. The TBDPS-substituted furan failed to react. Two alkynylsilanes were synthesized, but they also failed to react.     

The cell-penetrating peptide TAT(48-60) induces a non-lamellar phase in DMPC membranes.

Cell-penetrating peptides (CPPs) are short polycationic sequences that can translocate into cells without disintegrating the plasma membrane. CPPs are useful tools for delivering cargo, but their molecular mechanism of crossing the lipid bilayer remains unclear. Here we study the interaction of the HIV-derived CPP TAT (48-60) with model membranes by solid-state NMR spectroscopy and electron microscopy. The peptide induces a pronounced isotropic (31)P NMR signal in zwitterionic DMPC, but not in anionic DMPG bilayers. Octaarginine and to a lesser extent octalysine have the same effect, in contrast to other cationic amphiphilic membrane-active peptides. The observed non-lamellar lipid morphology is attributed to specific interactions of polycationic peptides with phosphocholine head groups, rather than to electrostatic interactions. Freeze-fracture electron microscopy indicates that TAT(48-60) induces the formation of rodlike, presumably inverted micelles in DMPC, which may represent intermediates during the translocation across eukaryotic membranes.