Thermolysis of coenzymes B12 at physiological temperatures: activation parameters for cobalt-carbon bond homolysis and a quantitative analysis of the perturbation of the homolysis equilibrium by the ribonucleoside triphosphate reductase from Lactobacillus leichmannii

The kinetics of the thermolysis of 5'-deoxyadenosylcobalamin (AdoCbl, coenzyme B12) in aqueous solution, pH 7.5, have been studied in the temperature range 30-85 degrees C using AdoCbl tritiated at the adenine C2 position and the method of initial rates. Combined with a careful analysis of the distribution of adenine-containing products, the results permit the dissection of the competing rate constants for carbon-cobalt bond homolysis and heterolysis. After correction for the temperature-dependent occurrence of the much less reactive base-off species of AdoCbl, the activation parameters for homolysis of the base-on species were found to be delta H++homo,on = 33.8 +/- 0.2 kcal mol-1 and delta S++homo,on = 13.5 +/- 0.7 cal mol-1 K-1, values not significantly different from those determined by Hay and Finke (J. Am. Chem. Soc. 108 (1986) 4820), in the temperature range 85-115 degrees C. In contrast, the heterolysis of base-on AdoCbl was characterized by a much smaller enthalpy of activation (delta H++het,on = 18.5 +/- 0.2 kcal mol-1) and a negative entropy of activation (delta S++het,on = -34.0 +/- 0.7 cal mol-1 K-1) so that heterolysis, which is minor pathway at elevated temperatures, is the dominant pathway for AdoCbl decomposition at physiological temperatures. Using literature values for the rate constant for the reverse reaction, the equilibrium constant for AdoCbl homolysis at 37 degrees C was calculated to be 7.9 x 10(-18). Comparison with the equilibrium constant for this homolysis at the active site of the ribonucleoside triphosphate reductase from Lactobacillus leichmannii shows that the enzymes shifts the equilibrium constant towards homolysis products by a factor of 2.9 x 10(12) (17.7 kcal mol-1) by binding the thermolysis products with an equilibrium constant of 7.1 x 10(16) M-2, compared to the bonding constant for AdoCbl of 2.4 x 10(4) M-1.     

Molecular modeling of the mechanochemical triggering mechanism for catalysis of carbon-cobalt bond homolysis in coenzyme B12

The possible contributions of the mechanochemical triggering effect to the enzymatic activation of the carbon-cobalt bond of coenzyme B12 (5'-deoxyadenosylcobalamin, AdoCbl) for homolytic cleavage have been studied by molecular modeling and semiempirical molecular orbital calculations. Classically, this effect has envisioned enzymatic compression of the axial Co-N bond in the ground state to cause upward folding of the corrin ring and subsequent sterically induced distortion of the Co-C bond leading to its destabilization. The models of this process show that in both methylcobalamin (CH3Cbl) and AdoCbl, compression of the axial Co-N bond does engender upward folding of the corrin ring, and that the extent of such upward folding is smaller in an analog in which the normal 5,6-dimethylbenzimidazole axial ligand is replaced by the sterically smaller ligand, imidazole (CH3(lm)Cbl and Ado(lm)Cbl). Furthermore, in AdoCbl, this upward folding of the corrin is accompanied by increases in the carbon-cobalt bond length and in the Co-C-C bond angle (which are also less pronounced in Ado(Im)Cbl), and which indicate that the Co-C bond is indeed destabilized by this mechanism. However, these effects on the Co-C bond are small, and destabilization of this bond by this mechanism is unlikely to contribute more than ca. 3 kcal mol(-1) towards the enzymatic catalysis of Co-C bond homolysis, far short of the observed ca. 14 kcal mol(-1). A second version of mechanochemical triggering, in which compression of the axial Co-N bond in the transition state for Co-C bond homolysis stabilizes the transition state by increased Co-N orbital overlap, has also been investigated. Stretching the Co-C bond to simulate the approach to the transition state was found to result in an upward folding of the corrin ring, a slight decrease in the axial Co-N bond length, a slight displacement of the metal atom from the plane of the equatorial nitrogens towards the "lower" axial ligand, and a decrease in strain energy amounting to about 8 kcal mol(-1) for both AdoCbl and Ado(Im)Cbl. In such modeled transition states, compression of the axial Co-N bond to just below 2.0 A (the distance subsequently found to provide maximal stabilization of the transition state by increased orbital overlap) required about 4 kcal mol(-1) for AdoCbl, and about 2.5 kcal mol(-1) for Ado(Im)Cbl. ZINDO/1 calculations on slightly simplified structures showed that maximal electronic stabilization of the transition state by about 10 kcal mol(-1) occurred at an axial Co-N bond distance of 1.96 A for both AdoCbl and Ado(Im)Cbl. The net result is that this type of transition state mechanochemical triggering can provide 14 kcal mol(-1) of transition state stabilization for AdoCbl, and about 15.5 kcal mol(-1) for the Ado(Im)Cbl, enough to completely explain the observed enzymatic catalysis. These results are discussed in the light of current knowledge about class I AdoCbl-dependent enzymes, in which the coenzyme is bound in its "base-off" conformation, with the lower axial ligand position occupied by the imidazole moiety of an active site histidine residue, and the class II enzymes, in which AdoCbl binds to the enzyme in its "base-on" conformation, and the pendent 5,6-dimethylbenzimidazole base remains coordinated to the metal during Co-C bond activation.     

Roles of β-d-ribofuranose ring and the functional groups of the d-ribose moiety of adenosylcobalamin in the diol dehydratase reaction

Four analogs of adenosylcobalamin (AdoCbl) modified in the d-ribose moiety of the Coβ ligand were synthesized, and their coenzyme properties were studied with diol dehydratase of Klebsiella pneumoniae ATCC 8724. 2′-Deoxyadenosylcobalamin (2′-dAdoCbl) and 3′-deoxyadenosylcobalamin (3′-dAdoCbl) were active as coenzyme. 2′,3′-Secoadenosylcobalamin (2′,3′-secoAdoCbl), an analog bearing the same functional groups as AdoCbl but nicked between the 2′ and 3′ in the ribose moiety, and its 2′,3′-dialdehyde derivative (2′,3′-secoAdoCbl dialdehyde) were totally inactive analogs of the coenzyme. It is therefore evident that the β-d-ribofuranose ring itself, possibly its rigid structure, is essential and much more important than the functional groups of the ribose moiety for coenzyme function (relative importance; β-d-ribofuranose ring ⪢ 3′-OH ⪢ 2′-OH ⪢ ether group). With 2′-dAloCbl and 3′-dAdoCbl as enzymes. an absorption peak at 478 nm appeared during enzymatic reaction, suggesting homolysis of the CCo bound to form cob(II)alamin as intermediate. In the absence of substrate, the complexes of the enzyme with these active analogs underwent rapid inactivation by oxygen. This suggests that their CCo bond is activated even in the absence of substrate by binding to the apoprotein. No significant spectral changes were observed with 2′,3′-secoAdoCbl upon binding to the apoenzyme. In contrast, spectroscopic observation indicates that 2′,3′-secoAdoCbl dialdehyde, another inactive analog, underwent gradual and irreversible cleavage of the CCo bond by interaction with the apodiol dehydratase, forming the enzyme-bound cob(II)alamin without intermediates.     

Hydrogen abstraction from thiols by adenosyl radicals: chemical precedent for thiyl radical formation, the first catalytic step in ribonucleoside triphosphate reductase from Lactobacillus leichmannii

Aqueous solutions of adenosylcobalamin (AdoCbl) were thermolyzed with excess beta-mercaptoethanol under anaerobic conditions. The product studies reveal that approximately 90% Co-C bond homolysis occurs, to yield Co(II)cobalamin, 5'-deoxyadenosine, and the disulfide product from the combination of two HOCH2CH2S* radicals, 2,2'-dithiodiethanol; there is also approximately 10% Co-C bond heterolysis, yielding Co(III)cobalamin, adenine, and 2,3-dihydroxy-4-pentenal. The kinetic studies show there is a first-order dependence on AdoCbl and zero-order dependence on thiol under the higher [RSH] experimental conditions used, consistent with the rate-determining step at high [RSH] being the generation of Ado*. The kinetic results require that, in enzyme-free AdoCbl solution, adenosyl radical (Ado*) is formed as a discrete intermediate which then abstracts H* from the added thiol. The activation parameters for Co-C bond homolysis in the presence of thiol trap are the same within experimental error as the activation parameters for Co-C bond homolysis without trap, standard delta H(obs) = 29(2) kcal mol(-1) and standard delta S(obs) = -1(5) e.u. The results, in comparison to the rate of Co-C bond homolysis in ribonucleoside triphosphate reductase (RTPR), reveal that RTPR accelerates Co-C bond cleavage in AdoCbl by approximately 10(10+/-1). The recent literature evidence bearing on the exact mechanism of RTPR enzymic cleavage of the Co-C bond of AdoCbl is briefly discussed, notably the fact that this mechanism is presently controversial, but does involve at least coupled (and possibly concerted) Co-C cleavage, -S-H cleavage, and C-H (Ado-H) formation steps.     

The synthesis of adenine-modified analogs of adenosylcobalamin and their coenzymic function in the reaction catalyzed by diol dehydrase

Five analogs of adenosylcobalamin modified in the adenine moiety of the Co beta ligand were synthesized and tested for coenzymic function with diol dehydrase of Klebsiella pneumoniae ATCC 8724. 1-Deaza and 3-deaza analogs of adenosylcobalamin were active as coenzyme, whereas 7-deaza and N6,N6-dimethyl derivatives and guanosylcobalamin did not show detectable coenzymic activity. 7-Deaza and N6,N6-dimethyl analogs acted as strong competitive inhibitors with respect to adenosylcobalamin. The formation of cob(II)alamin as intermediate in the catalytic reaction was spectroscopically observed with catalytically active complexes of the enzyme with 1-deaza and 3-deaza analogs in the presence of 1,2-propanediol, but not with complexes with the inactive analogs. Oxygen sensitivity of the enzyme-analog complexes suggests that the carbon-cobalt bond of 1-deaza and 3-deaza analogs becomes activated by the enzyme even in the absence of substrate. These results indicate that the importance of the nitrogen atoms in the adenine moiety of the coenzyme for manifestation of catalytic function and for activation of the carbon-cobalt bond decreases in the following order: N-7 greater than 6-NH2 greater than N-3 greater than N-1. The dissociation constant for 5'-deoxyadenosine determined by equilibrium dialysis at 37 degrees C was about 23 microM.     

9-(Adenylyl)alkylcobalamins as inhibitors of adenosylcobalamin-dependent glycerol dehydratase from Aerobacter aerogenes

The behavior of two coenzyme analogs, [(5-aden-9-yl)methoxyethyl] cob (III) alamin and [(5-aden-9-yl)pentyl] cob (III) alamin modified at the nucleoside ligand sugar moiety was studied in the system of adenosyl-cobalamin-dependent glycerol dehydratase from Aerobacter aerogenes. It was shown that neither of the analogs possesses coenzyme properties and that both are strong competitive inhibitors for adenosylcobalamin (AdoCbl). The affinity of the two analogs for the apoenzyme is higher than that of AdoCbl. The data obtained are indicative of the essential role of the ribofuranoside fragment of AdoCbl in the manifestation of the coenzyme activity. The apoenzyme interaction with the analogs under study is discussed in terms of the Dreiding stereomodels for AdoCbl and its analogs.     

Coenzyme AdoB12 vs AdoB12.-homolytic Co-C cleavage following electron transfer: a rate enhancement greater than or equal to 10(12)

Comparison of the 25 degrees C Co-C bond homolysis rate constant of adenosyl-cobalamin (coenzyme B12) vs that for electrochemically reduced adenosyl-cobalamin radical anion indicates a rate enhancement of at least 10(12 +/- 2) upon the addition of one antibonding electron. Even though electrochemical reduction promotes Co-C homolysis by virtually the same amount as the 10(12 +/- 1) enzymic activation seen for adenosylcobalamin, electron-transfer activation of the Co-C homolysis in adenosylcobalamin-dependent enzyme reactions is considered unlikely, based on four lines of evidence.     

The mechanism of adenosylcobalamin-dependent rearrangements

Adenosylcobalamin (AdoCbl)-dependent rearrangements are a group of reactions with no obvious precedents in organic chemistry. In every case, they are characterized by a mechanism in which a hydrogen atom on one carbon atom exchanges places with a group X on an adjacent carbon: (formula; see text) Much experimental work indicates that an AdoCbl rearrangement is initiated by homolysis of the C-Co bond of the cofactor. The migrating hydrogen is then abstracted from the substrate by the resulting 5'-deoxyadenosyl radical, or by a second radical that is generated elsewhere at the active site, and, after the migration of group X, is returned to the product in a similar reaction. In at least some of the rearrangements, group X migration may occur via a cation radical intermediate that formed by the departure of X with its electrons, a process assisted by the unpaired electron left behind on the adjacent carbon after the abstraction of the migrating hydrogen. Once C-Co bond cleavage has initiated the reaction by producing a free radical at the active site, the corrin ring plays no further role in the rearrangements.     

Carbon-13 nuclear magnetic resonance studies of adenosylcobalamin and alkylcorrinoids, selectively enriched with carbon-13.

The carbon-13 nuclear magnetic resonance spectra of a series of alkylcorrinoids, selectively enriched with 13C in the alkyl ligand, were recorded at 25.2 MHz and 25 degrees. The nature of the axial ligands markedly affects the chemical shift of the labeled alkyl moiety (trans effect) as well as the 13C resonances of selected carbon atoms of the corrin ring (cis effect). Although a number of factors appear to influence the trans effect on the chemical shift of the alkyl ligand (important among them being electric field effects), the cis effect appears to be dominated by changes in charge density (at the methine bridge carbon atoms, C-5, C-10, C-15) and by steric effects (at the methyl groups at C-1, C-5, and C-15) accompanying axial ligation. Spin-latice relaxation times of several organocorrinoids, selectively labeled with 13C in the ligands attached to cobalt, were also measured. The T1 values of the methylene carbons of [5'-13C]adenosylcobalamin and [2-13C]carboxymethylcobalamin are very similar to that of the methine bridge carbon atom C-10 of the corrin ring, indicating that rotation about the carbon-cobalt bond of these two corrinoids is severely restricted. On the other hand, internal rotation about the carbon-cobalt bond of methylcobalamin is rapid.     

The synthesis and properties of four spin-labeled analogs of adenosylcobalamin

Four spin-labeled analogs of adenosylcobalamin have been synthesized to aid in the detection and identification of radical intermediates in the adenosylcobalamin-dependent enzymatic reactions and to serve as probes of the coenzyme, substrate, and effector binding sites of the protein. Three isomers of adenosylcobalamin, in which one of the propionamide side chains (b, d, or e) was hydrolyzed, and adenosylepicobalamin e-carboxylic acid were reacted with 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to yield the spin-labeled adenosylcorrinoids. These spin-labeled derivatives of adenosylcobalamin function as coenzymes and/or inhibitors of dioldehydrase from Klebsiella pneumoniae and of ribonucleotide reductase from Corynebacterium nephridii. Electron spin resonance has been used to monitor the photolytic cleavage of the carbon-cobalt bond of these analogs.     

Substrate-induced conformational change of a coenzyme B12-dependent enzyme: crystal structure of the substrate-free form of diol dehydratase

Substrate binding triggers catalytic radical formation through the cobalt-carbon bond homolysis in coenzyme B12-dependent enzymes. We have determined the crystal structure of the substrate-free form of Klebsiella oxytoca diol dehydratase*cyanocobalamin complex at 1.85 A resolution. The structure contains two units of the heterotrimer consisting of alpha, beta, and gamma subunits. As compared with the structure of its substrate-bound form, the beta subunits are tilted by approximately 3 degrees and cobalamin is also tilted so that pyrrole rings A and D are significantly lifted up toward the substrate-binding site, whereas pyrrole rings B and C are only slightly lifted up. The structure revealed that the potassium ion in the substrate-binding site of the substrate-free enzyme is also heptacoordinated; that is, two oxygen atoms of two water molecules coordinate to it instead of the substrate hydroxyls. A modeling study in which the structures of both the cobalamin moiety and the adenine ring of the coenzyme were superimposed onto those of the enzyme-bound cyanocobalamin and the adenine ring-binding pocket, respectively, demonstrated that the distortions of the Co-C bond in the substrate-free form are already marked but slightly smaller than those in the substrate-bound form. It was thus strongly suggested that the Co-C bond becomes largely activated (labilized) when the coenzyme binds to the apoenzyme even in the absence of substrate and undergoes homolysis through the substrate-induced conformational changes of the enzyme. Kinetic coupling of Co-C bond homolysis with hydrogen abstraction from the substrate shifts the equilibrium to dissociation.     

Electronic structure studies of the adenosylcobalamin cofactor in glutamate mutase

Glutamate mutase (GM) is a cobalamin-dependent enzyme that catalyzes the reversible interconversion of L-glutamate and L-threo-3-methylaspartate via a radical-based mechanism. To initiate catalysis, the 5'-deoxyadenosylcobalamin (AdoCbl) cofactor's Co-C bond is cleaved homolytically to generate an adenosyl radical and Co2+ Cbl. In this work, we employed a combination of spectroscopic and computational tools to evaluate possible mechanisms by which the Co-C bond is activated for homolysis. Minimal perturbations to the electronic absorption (Abs), circular dichroism (CD), and magnetic CD (MCD) spectra of AdoCbl are observed upon formation of holoenzyme, even in the presence of substrate (or a substrate analogue), indicating that destabilization of the Co3+ Cbl "ground state" is an unlikely mechanism for Co-C bond activation. In contrast, striking alterations are observed in the spectroscopic data of the post-homolysis product Co2+ Cbl when bound to glutamate mutase in the presence of substrate (or a substrate analogue) as compared to unbound Co2+ Cbl. These enzymatic perturbations appear to most strongly affect the metal-to-ligand charge-transfer transitions of Co2+ Cbl, suggesting that the cofactor/active-site interactions give rise to a fairly uniform stabilization of the Co 3d orbitals. Remarkable similarities between the results obtained in this study and those reported previously for the related Cbl-dependent isomerase methylmalonyl-CoA mutase indicate that a common mechanism by which the cofactor's Co-C bond is activated for homolytic cleavage may be operative for all base-off/His-on Cbl-dependent isomerases.     

Theoretical evaluation of the hydrogen kinetic isotope effect on the first step of the methylmalonyl-CoA mutase reaction

We have calculated hydrogen kinetic isotope effects (KIEs) for the first step of the methylmalonyl-CoA mutase reaction, including multidimensional tunneling correction at the zero curvature (ZCT) level, and compared them with the experimental values. Both alternative mechanisms of this step, concerted and stepwise, can be accommodated. It turned out to be essential to include Arg207 hydrogen-bonded to the reactant in the mechanism predicting simultaneous breaking of the Co-C bond of AdoCbl and hydrogen atom transfer. The consequence of the stepwise mechanism is a much larger facilitation of the homolytic dissociation of the carbon-cobalt bond by the enzyme than currently appreciated; our results suggest lowering of the activation energy by about 23 kcal mol(-1). We have also shown that large hydrogen KIEs of tunneling origin do not necessarily break the Swain-Schaad equation. Furthermore, when this equation does not hold, the exponent may be smaller in the presence of tunneling than it is at the semi-classical limit, indicating that nonclassical behavior may be a more common phenomenon than expected.     

Importance of the histidine ligand to coenzyme B12 in the reaction catalyzed by methylmalonyl-CoA mutase

Methylmalonyl-CoA mutase is an adenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA. The crystal structure of this protein revealed that binding of the cofactor is accompanied by a significant conformational change in which dimethylbenzimidazole, the lower axial ligand to the cobalt in solution, is replaced by His-610 donated by the active site. The contribution of the lower axial base to the approximately 10(12)-fold rate acceleration of the homolytic cleavage of the upper axial cobalt-carbon bond has been the subject of intense scrutiny in the model inorganic literature. In contrast, trans ligand effects in methylmalonyl-CoA mutase and indeed the significance of the ligand replacement are poorly understood. In this study, we have used site-directed mutagenesis to create the H610A and H610N variants of methylmalonyl-CoA mutase and report that both mutations exhibit both diminished activity (5,000- and 40,000-fold, respectively) and profoundly weakened affinity for the native cofactor, AdoCbl. In contrast, binding of the truncated cofactor analog, adenosylcobinamide, lacking the nucleotide tail, is less impaired. The catalytic failure of the His-610 mutants is in marked contrast to the phenotype of the adenosylcobinamide-GDP reconstituted wild type enzyme that exhibits only a 4-fold decrease in activity, although His-610 fails to coordinate when this cofactor analog is bound. Together, these studies suggest that His-610 may: (i) play a structural role in organizing a high affinity cofactor binding site possibly via electrostatic interactions with Asp-608 and Lys-604, as suggested by the crystal structure and (ii) play a role in catalyzing the displacement of dimethylbenzimidazole thereby facilitating the conformational change that must precede cofactor docking to the mutase active site.     

The coenzyme b12 analog 5'-deoxyadenosylcobinamide-gdp supports catalysis by methylmalonyl-coa mutase in the absence of trans-ligand coordination

Methylmalonyl-CoA mutase is an 5'-adenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA. The crystal structure of this protein revealed that binding of the cofactor is accompanied by a significant conformational change in which dimethylbenzimidazole, the lower axial ligand to cobalt in solution, is replaced by His(610) donated by the active site. The role of the lower axial ligand in the trillion-fold labilization of the upper axial cobalt-carbon bond has been the subject of enduring debate in the model inorganic literature. In this study, we have used a cofactor analog, 5'deoxyadenosylcobinamide GDP (AdoCbi-GDP), which reconstitutes the enzyme in a "histidine-off" form and which allows us to evaluate the contribution of the lower axial ligand to catalysis. The k(cat) for the enzyme in the presence of AdoCbi-GDP is reduced by a factor of 4 compared with the native cofactor AdoCbl. The overall deuterium isotope effect in the presence of AdoCbi-GDP ((D)V = 7.2 +/- 0.8) is comparable with that observed in the presence of AdoCbl (5.0 +/- 0.6) and indicates that the hydrogen transfer steps in this reaction are not significantly affected by the change in coordination state of the bound cofactor. These surprising results are in marked contrast to the effects ascribed to the corresponding lower axial histidine ligands in the cobalamin-dependent enzymes glutamate mutase and methionine synthase.     

Examination of structural characteristics of the potent oxytocin antagonists [dPen1,Pen6]-OT and [dPen1,Pen6, 5-tBuPro7]-OT by NMR, Raman, CD spectroscopy and molecular modeling.

The synthesis and biological evaluation of penicillamine(6)-5-tert-butylproline(7)-oxytocin analogs and comparison with their proline(7)-oxytocin counterparts has led to the discovery of two potent oxytocin (OT) antagonists: [dPen(1),Pen(6)]-oxytocin (1, pA(2) = 8.22, EC(50) = 6.0 nM) and [dPen(1),Pen(6),5-tBuPro(7)]-oxytocin (2, pA(2) = 8.19, EC(50) = 6.5 nM). In an attempt to understand the conformational requirements for their biological activity, spectroscopic analyses of 1 and 2 were performed using (1)H NMR, laser Raman and CD techniques. In H(2)O, oxytocin analogs 1 and 2 exhibited cis-isomer populations of 7% and 35%, respectively. Measurement of the amide proton temperature coefficients revealed solvent shielded hydrogens for Gln(4) and Pen(6) in the major trans-conformer of 1 as well as for Gln(4) in the minor cis-conformer of 2. Few long-distance NOEs were observed, suggesting conformational averaging for analogs 1 and 2 in water; moreover, a lower barrier (16.6 +/- 0.2 kcal/mol) for isomerization of the amide N-terminal to 5-tBuPro(7) relative to OT was calculated from measuring the coalescence temperature of the Gly(9) backbone NH signals in the NMR spectra of 2. Observed bands in the Raman spectra of 1 and 2 correspond to C(beta)-S-S-C(beta) dihedral angles of +110-115 degrees and +/-90 degrees , respectively. In water, acetonitrile and methanol, the CD spectra for 1 exhibited a positive maximum around 236-239 nm; in trifluoroethanol, the spectra shifted and a negative maximum was observed at 240 nm. The CD spectra of 2 were unaffected by solvent changes and exhibited a negative maximum at 236-239 nm. The CD and Raman data both suggested that a conformation having a right-handed screw sense about the disulfide and a chi(CS-SC) dihedral angle value close to 115 degrees was favored for analog 1 in water, methanol and acetonitrile, but not trifluoroethanol, where a +/-90 degrees angle was favored. Analog 2 was more resilient to conformational change about the disulfide, and adopted a preferred disulfide geometry corresponding to a +/-90 degrees chi(CS-SC) dihedral angle. Monte Carlo conformational analysis of analogs 1 and 2 using distance restraints derived from NMR spectroscopy revealed two prominent conformational minima for analog 1 with disulfide geometries around +114 degrees and +116 degrees . Similar analysis of analog 2 revealed one conformational minimum with a disulfide geometry around +104 degrees . In sum, the conformation about the disulfide in [dPen(1),Pen(6)]-OT (1) was shown to be contingent on environment and in TFE, adopted a geometry similar to that of [dPen(1),Pen(6),5-tBuPro(7)]-OT (2) which appeared to be stabilized by hydrophobic interactions between the 5-tBuPro(7) (5R)-tert-butyl group, the Leu(8) isopropyl sidechain and the Pen(6)beta-methyl substituents. In light of the conformational rigidity of 2 about the disulfide bond, and the similar geometry adopted by 1 in TFE, a S-S dihedral angle close to +110 degrees may be a prerequisite for their binding at the receptor.     

Mechanism of radical-based catalysis in the reaction catalyzed by adenosylcobalamin-dependent ornithine 4,5-aminomutase

We report an analysis of the reaction mechanism of ornithine 4,5-aminomutase, an adenosylcobalamin (AdoCbl)- and pyridoxal L-phosphate (PLP)-dependent enzyme that catalyzes the 1,2-rearrangement of the terminal amino group of D-ornithine to generate (2R,4S)-2,4-diaminopentanoic acid. We show by stopped-flow absorbance studies that binding of the substrate D-ornithine or the substrate analogue D-2,4-diaminobutryic acid (DAB) induces rapid homolysis of the AdoCbl Co-C bond (781 s(-1), D-ornithine; 513 s(-1), DAB). However, only DAB results in the stable formation of a cob(II)alamin species. EPR spectra of DAB and [2,4,4-(2)H(3)]DAB bound to holo-ornithine 4,5-aminomutase suggests strong electronic coupling between cob(II)alamin and a radical form of the substrate analog. Loading of substrate/analogue onto PLP (i.e. formation of an external aldimine) is also rapid (532 s(-1), D-ornithine; 488 s(-1), DAB). In AdoCbl-depleted enzyme, formation of the external aldimine occurs over long time scales (approximately 50 s) and occurs in three resolvable kinetic phases, identifying four distinct spectral intermediates (termed A-D). We infer that these represent the internal aldimine (lambda(max) 416 nm; A), two different unliganded PLP states of the enzyme (lambda(max) at 409 nm; B and C), and the external aldimine (lambda(max) 426 nm; D). An imine linkage with d-ornithine and DAB generates both tautomeric forms of the external aldimine, but with D-ornithine the equilibrium is shifted toward the ketoimine state. The influence of this equilibrium distribution of prototropic isomers in driving homolysis and stabilizing radical intermediate states is discussed. Our work provides the first detailed analysis of radical-based catalysis in this Class III AdoCbl-dependent enzyme.     

Co-C force constants from resonance Raman spectra of alkylcobalamins: insensitivity to dimethylbenzylimidazole coordination

The Co-C stretching vibration has been identified in resonance Raman spectra of alkyl-cobalamins, via isotope substitution, permitting estimation of the Co-C force constants, f = 1.85, 1.77 and 1.50 mdyn Å⁻¹ for methyl-, ethyl- and deoxyadenosyl-cobalamin, respectively (ν_Co-C = 506, 471 and 442/429 cm⁻¹). These values scale with the reported bond dissociation energies, and support the view that the Co-C bond weakens with increasing bulk of the alkyl group due to steric interaction with the corrin ring. However, the force constants are unaffected by dissociation of the dimethylbenzimidazole ligand at low pH, even though the bond dissociation energy rises significantly upon DMB dissociation in AdoCbl. This increase must therefore reflect destabilization of the Co^II product, rather than Co-C bond strengthening in the AdoCbl ground state. The insensitivity of the force constants to dimethylbenzimidazole dissociation implies that the steric effect of DMB coordination is not transmitted to the Co-C bond by the corrin ring. Consistent with this interpretation, the RR frequencies of the corrin ring modes are minimally perturbed by DMB dissociation, supporting earlier NMR results that indicated little change in the corrin conformation.     

Conformational stability and activity of ribonuclease T1 with zero, one, and two intact disulfide bonds

Ribonuclease T1 has two disulfide bonds linking cysteine residues 2-10 and 6-103. We have prepared a derivative of ribonuclease T1 in which one disulfide bond is broken and the cysteine residues carboxymethylated, (2-10)-RCM-T1, and three derivatives in which both disulfides are broken and the cysteine residues reduced, R-T1, carboxamidomethylated, RCAM-T1, or carboxymethylated, RCM-T1. The RNA hydrolyzing activity of these proteins has been measured, and urea and thermal denaturation studies have been used to determine conformational stability. The activity, melting temperature, and conformational stability of the proteins are: ribonuclease T1 (100%, 59.3 degrees C, 10.2 kcal/mol), (2-10)-RCM-T1 (86%, 53.3 degrees C, 6.8 kcal/mol), R-T1 (53%, 27.2 degrees C, 3.0 kcal/mol), RCAM-T1 (43%, 21.2 degrees C, 1.5 kcal/mol), and RCM-T1 (35%, 16.6 degrees C, 0.9 kcal/mol). Thus, the conformational stability is decreased by 3.4 kcal/mol when one disulfide bond is broken and by 7.2-9.3 kcal/mol when both disulfide bonds are broken. It is quite remarkable that RNase T1 can fold and function with both disulfide bonds broken and the cysteine residues carboxymethylated. The large decrease in the stability is due mainly to an increase in the conformational entropy of the unfolded protein which results when the constraints of the disulfide bonds on the flexibility are removed. We propose a new equation for predicting the effect of a cross-link on the conformational entropy of a protein: delta Sconf = -2.1 - (3/2)R 1n n, where n is the number of residues between the side chains which are cross-linked. This equation gives much better agreement with experimental results than other forms of this equation which have been used previously.     

Highly active analogs of 1alpha,25-dihydroxyvitamin D(3) that resist metabolism through C-24 oxidation and C-3 epimerization pathways

The secosteroid hormone 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is metabolized in its target tissues through modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the main side chain modification pathway is initiated by hydroxylation at C-24 of the side chain and leads to the formation of the end product, calcitroic acid. The C-23 and C-26 oxidation pathways, the minor side chain modification pathways are initiated by hydroxylations at C-23 and C-26 of the side chain and lead to the formation of the end product, calcitriol lactone. The C-3 epimerization pathway, the newly discovered A-ring modification pathway is initiated by epimerization of the hydroxyl group at C-3 of the A-ring to form 1alpha,25(OH)(2)-3-epi-D(3). A rational design for the synthesis of potent analogs of 1alpha,25(OH)(2)D(3) is developed based on the knowledge of the various metabolic pathways of 1alpha,25(OH)(2)D(3). Structural modifications around the C-20 position, such as C-20 epimerization or introduction of the 16-double bond affect the configuration of the side chain. This results in the arrest of the C-24 hydroxylation initiated cascade of side chain modifications at the C-24 oxo stage, thus producing the stable C-24 oxo metabolites which are as active as their parent analogs. To prevent C-23 and C-24 hydroxylations, cis or trans double bonds, or a triple bond are incorporated in between C-23 and C-24. To prevent C-26 hydroxylation, the hydrogens on these carbons are replaced with fluorines. Furthermore, testing the metabolic fate of the various analogs with modifications of the A-ring, it was found that the rate of C-3 epimerization of 5,6-trans or 19-nor analogs is decreased to a significant extent. Assembly of all these protective structural modifications in single molecules has then produced the most active vitamin D(3) analogs 1alpha,25(OH)(2)-16,23-E-diene-26,27-hexafluoro-19-nor-D(3) (Ro 25-9022), 1alpha,25(OH)(2)-16,23-Z-diene-26,27-hexafluoro-19-nor-D(3) (Ro 26-2198), and 1alpha,25(OH)(2)-16-ene-23-yne-26,27-hexafluoro-19-nor-D(3) (Ro 25-6760), as indicated by their antiproliferative activities.