Solvent and substrate deuterium isotope effects on a transamination reaction catalyzed by pig heart aspartate aminotransferase.

Transamination of erythro-β-hydroxy-l-aspartate catalyzed by pig heart aspartate aminotransferase (EC 2.6.1.1) was studied with both normal and α-deuterated substrate in $\text{H}{}_2\text{O}\text{D}{}_2\text{O}$. The overall transamination reaction, with α-ketoglutarate as amino group acceptor, showed no primary substrate isotope effect. However, one of the elementary reactions between two enzyme-substrate complexes was found to exhibit large primary isotope effects in both the forward and the reverse directions. This same reaction also showed a twofold solvent isotope effect in the reverse direction, but D₂O had only a negligible effect in the forward direction. These data were interpreted to indicate that the substrate α-hydrogen arises from a Bronsted acid with two equivalent hydrogens. Another elementary reaction, which is 100-fold slower, was also studied since it appeared to be one of the principal rate-determining steps in the overall reaction. This step was not affected by substrate deuteration but exhibited large solvent isotope effects in both directions.     

Anomerization of glucose 6-phosphate: catalysis by phosphoglucose isomerase.

The anomerase (1-epimerase) activity of phosphoglucose isomerase (d-glucose 6-phosphate ketol-isomerase EC 5.3.1.9) has been studied. The pH-V_max profile, assayed by two different methods, shows a dependence on two ionizable groups in the enzyme with pK values of 7.0 and 9.3 at 0 °C. Additionally, an unusual reversal of the basic leg of the normal profile to yield a large increase in V_max is observed above pH 9.5. Deuterium solvent isotope effects of are observed for isomerase and anomerase activities respectively. An anomerase mechanism similar to noncatalyzed anomerization is postulated with a discussion of the catalytic groups involved.     

Anomerization of glucose 6-phosphate. pH dependence and solvent isotope effects.

The anomerization of α-d-glucose 6-phosphate has been examined using a spectrophotometric coupled enzyme assay. The pH-rate profile for spontaneous d-glucose 6-phosphate anomerization reveals that the d-glucose 6-phosphate dianion is the species giving rise to the much higher rate of d-glucose 6-phosphate anomerization over that of d-glucose. A deuterium solvent isotope effect of is consistent with the postulated intramolecular general-base catalysis by the phosphate.     

H/2H isotope effect in redox reactions of cytochrome c

The rate of reaction of ferro- and ferricytochrome c (C(II) and C(III)) with ferri- and ferrocyanide and of C(III) with O₂^? and CO₂^? was determined in H₂O and in ²H₂O in the temperature range 5–35 °C. No isotope effect was evident in any of the reductions of C(III); the apparent energy of activation was identical in H₂O and ²H₂O. An isotope effect with , depending on pH for instance was observed in the oxidation of C(II), in the slow phase of oxidation which involves conformational changes. An interpretation (supported by evidence from previous work) involving water molecules in the close vicinity of the reaction site on the protein is discussed.     

Additivity of isotope effects for successive deuteration in the deacetylation of acetyl-α-chymotrypsin

α-Chymotrypsin, converted to the acetyl enzyme by the p-nitrophenyl esters of CH₃COOH, CH₂DCOOH, CHD₂COOH, and CD₃COOH, undergoes deacetylation at pH 7.6 (phosphate buffer) and 25°C with secondary isotope effects of $\text{k(}\text{CH}{}_3\text{)}\text{k(}\text{CH}{}_2\text{D}\text{)}\text{= 0.985 ± 0.006}$, $\text{k(}\text{CH}{}_3\text{)}\text{k(}\text{CHD}{}_2\text{)}\text{= 0.971 ± 0.010}$, and $\text{k(}\text{CH}{}_3\text{)}\text{k(}\text{CD}{}_3\text{)}\text{= 0.956 ± 0.008}$. These isotope effects obey the simple additivity rule (“Rule of the Geometric Mean”) to within 20 J/mol, corresponding to about 5–6% of the maximum isotope effect for carbonyl addition. Thus, to this level, the three hydrogenic sites of the acetyl group are not rendered distinct in their contributions to the overall isotope effect even in the chiral environment of the chymotrypsin active site.     

Catalysis of ester hydrolysis by N-(2-dimethylaminoethyl)acetohydroxamic acid (I)

N-(2-dimethylaminoethyl)acetohydroxamic acid was synthesized. This compound, which incorporates a dimethylamino group as a second functionality into the hydroxamic acid molecule, catalyzes the hydrolysis of p-nitrophenyl acetate faster than acetohydroxamic acid itself does. The function of the dimethylamino group is to labilize the intermediate formed in the reaction, thus assisting deacylation intramolecularly. The dimethylamino group carries out this function by intramolecular general base catalysis. Nucleophilic catalysis is ruled out by the sizable deuterium oxide solvent isotope effect () found. General acid-hydroxide ion catalysis is ruled out by determination of the lack of reaction with azide ion, which does not possess a dissociable proton, with the intermediate in this reaction. The deuterium oxide solvent isotope effect on the azide ion reaction of the intermediate also rules out a general acid-hydroxide ion reaction.     

Kinetic alpha-deuterium isotope effects for enzymatic and nonenzymatic hydrolysis of nicotinamide-beta-riboside.

The kinetic α-secondary deuterium isotope effect, $\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}$, for the pH-independent hydrolysis of nicotinamide riboside, yielding nicotinamide and ribose, in water at 25 ° is 1.14, establishing that this reaction proceeds with unimolecular substrate decomposition to yield a carboxonium ion, or related species, in the rate-determining step. Surprisingly, the corresponding isotope effect for the base-catalyzed decomposition of the same substrate is 1.12, a value indicating considerable sp² character at the Cl′ position in the transition state for this reaction. A similar result, $\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}\text{= 1.15}$, was obtained for base-catalyzed hydrolysis of NAD⁺. The kinetic alpha deuterium isotope effect for the pig brain NAD glycohydrolasecatalyzed hydrolysis of nicotinamide riboside is 1.08. This value suggests that CN bond cleavage to form an intermediate carboxonium ion, or structurally related species, is at least partially rate-determining. In contrast, the corresponding value for the hydrolysis of this substrate catalyzed by Escherichia coli nicotinamide ribonucleotide glycohydrolase is very near unity, a result consistent with several interpretations including a rate-determining enzyme isomerization reaction.     

The dehalogenation of iodouracil by cysteine. Intramolecular general-acid catalysis of cysteine addition to 5-iodouracil

The rate-determining step of the cysteine-catalyzed deiodination of 5-iodouracil is the formation of 5-iodo-6-cysteinyl-5,6-dihydrouracil. The rate of the reaction depends upon the concentration of un-ionized 5-iodouracil and the following ionic species of cysteine; ^?OOC(NH₃⁺)CHCH₂S^?. Unlike the reaction of 2-mercapto-ethanol with 5-iodouracil, the cysteine reaction is not subject to catalysis by imidazolium ion and tris(hydroxymethyl)aminomethane hydrochloride. When the rates of cysteine reacting with 5-iodouracil are measured in both H₂O and D₂O, a large kinetic isotope effect is observed ($\text{k}{}_2{}^{\mathrm{<mtext>H</mtext><mtext>20</mtext>}}\text{k}{}_2{}^{\mathrm{<mtext>D</mtext><mtext>20</mtext>}}\text{= 4.10}$), thus implicating the protonated α amino group of cysteine as an intramolecular general acid catalyst for the reaction. These results and possible mechanisms for the actual dehalogenation of the intermediate 5-iodo-6-cysteinyl-5,6-dihydrouracil are discussed in terms of a possible mechanism for enzymatic halopyrimidine dehalogenation.     

Mechanistic studies of epoxide hydrolase utilizing a continuous spectophotometric assay

A precise continuous photometric assay has been devised and utilized for mechanistic studies of chicken and rat liver microsomal epoxide hydrolase (EH). The assay is based on monitoring the hydration of p-nitrostyrene oxide (PNSO) at 310 nm. Rat liver EH hydrates S-(+)- and R-(?)-PNSO differentially, the K_m and V values for the former being ca. four times those for the latter; in contrast, enantiomeric differences are negligible with chicken liver EH. With rat EH V increases slightly from pH 7 to 8 and then falls rapidly from pH 8 to 9.5; K_m remains constant from pH 7 to 8 and then increases steadily from pH 8 to 9.5. In 86 mol% D₂O the solvent isotope effect on V ($\text{H}{}_2\text{O}\text{D}{}_2\text{O}$) is 1.103 ± 0.015. Both rat and chicken EH show a 3% inverse isotope effect for the hydration of [7-²H]PNSO and a 4% normal isotope effect for the hydration of [8-²H₂]PNSO. These observations are discussed in terms of the possible participation of acid as well as base catalysis in the enzymatic mechanism.     

Intramolecular determination of primary kinetic isotope effects in hydroxylations catalyzed by cytochrome P-450

Isotope effects for hydroxylation reactions catalyzed by cytochrome P-450 have usually been measured by comparing the overall reaction velocities of deuterated and nondeuterated substrates. Since the rate-limiting step is probably not the single reaction involving covalent bond cleavage, such an approach does not yield information about the primary isotope effect. We measured the primary kinetic isotope effect for benzylic hydroxylation by a method utilizing intramolecular competition, using the symmetrical substrate 1,3-diphenylpropane-1,1-d₂. These experiments yield a value of $\text{k}{}_{\mathrm{<mtext>H</mtext>}}\text{k}{}_{\mathrm{D}}\text{= 11}$, a larger effect than has previously been reported for benzylic hydroxylations.     

Malate dehydrogenase. Kinetic studies with meso-tartrate and 2-keto-3-hydroxysuccinate, comparison of the mitochondrial and supernatant pig heart enzymes.

Initial rate, product inhibition, and isotope rate kinetic studies of pig heart mitochondrial and supernatant malate dehydrogenases, acting upon the nonphysiological substrates, meso-tartrate and 2-keto-3-hydroxysuccinate, are reported. The measured spontaneous keto-enol equilibrium for 2-keto-3-hydroxysuccinate in 0.05 m Tris-acetate (pH 8.0) at 25 °C favors the enol form, dihydroxyfumarate, with an apparent equilibrium constant of 0.036. The enzyme-catalyzed reaction favors meso-tartrate with an apparent equilibrium constant of 1.25 × 10^?6, M^?1 at pH 8.0. The mechanism apparently remains ordered bi bi for both enzymes when these nonphysiological substrates are used, and the chemical-converting hydride transfer step becomes more rate limiting for both enzymes. This conclusion is supported by $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{V}{}_{\mathrm{<mtext>D</mtext>}}$ and $\text{(}\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{K}{}_{\mathrm{<mtext>H</mtext>}}\text{)}\text{V}{}_{\mathrm{<mtext>D</mtext>}}\text{K}{}_{\mathrm{<mtext>D</mtext>}}$ values of 2.6 and 3.1, respectively, for the mitochondrial enzyme and 1.9 and 2.9, respectively, for the supernatant enzyme.     

Hydration of Escherichia coli lipids: Deuterium T1 relaxation time studies of phosphatidylglycerol,phosphatidylethanolamine and phosphatidylcholine

The hydration properties of Escherichia coli lipids (phosphatidylglycerol, phosphatidylethanolamine) and synthetic $\text{1,2-}\text{dioleoyl}\text{-sn-}\text{glycero-3-phosphocholine}$ in H₂O/²H₂O mixtures (9:1, v/v) were investigated with ²H-NMR. Comparison of the ²H₂O spin lattice relaxation time ($\text{T}{}_1$) as a function of the water content revealed a remarkable quantitative similarity of all three lipid-H₂O systems. Two distinct hydration regions could be discerned in the $\text{T}{}_1$ relaxation time profile. (1) A minimum of 11–16 water molecules was needed to form a primary hydration shell, characterized by an average relaxation time of $\text{T}{}_1\text{≈ 90}\text{ms}$. (2) Additional water was found to be in exchange with the primary hydration shell. The exchange process could be described in terms of a two-site exchange model, assuming rapid exchange between bulk water with $\text{T}{}_1\text{= 500}\text{ms}$ and hydration water with $\text{T}{}_1\text{= 80–120}\text{ms}$. Analysis of the linewidth and the residual quadrupole splitting (at low water content) confirmed the size of the primary hydration layer. However, each lipid-water system exhibited a somewhat different linewidth behavior, and a detailed molecular interpretation appeared to be preposterous.     

Influence of ionic strength upon the proton inventory for the water-catalyzed hydrolysis of N-acetylbenzotriazole

Proton inventory investigations of the hydrolysis N-acetylbenzotriazole at pH 3.0 (or the equivalent point on the pD rate profile) have been conducted at two different temperatures and at ionic strengths ranging from 0 to 3.0 M. The solvent deuterium isotope effects and proton inventories are remarkably similar over this wide range of conditions. The proton inventories suggest a cyclic transition state involving four protons contributing to the solvent deuterium isotope effect for the water-catalyzed hydrolysis. The hydrolysis data are described by the equation $\text{k}{}_{\mathrm{n}}\text{= k}{}_{\mathrm{<mtext>o</mtext>}}\text{(1 ? n + nπ}{}_{\mathrm{<mtext>a</mtext>}}{}^1\text{)}{}^4$ with $\text{π}{}_{\mathrm{<mtext>a</mtext>}}{}^1\text{～ 0.74}$, where k_o is the observed first-order rate constant in protium oxide, n is the atom fraction of deuterium in the solvent, k_n is the rate constant in a protium oxide-deuterium oxide mixture, and $\text{π}{}_{\mathrm{<mtext>a</mtext>}}{}^1$ is the isotopic fractionation factor.     

The addition of bisulfite to 5-fluorouracil. Evidence for a change in rate determining step

The kinetics of bisulfite addition to 5-fluorouracil were studied as a function of increasing concentrations of potential general acids. Values of $\text{k}{}_{\mathrm{obsd}}\text{[}\text{SO}{}_3{}^{\mathrm{=}}\text{]}$ measured at 25°C and ionic strength 1.0 M increased linearly and then became invariant with increasing concentrations of either HSO₃^? or (OHCH₂CH₂)₂N⁺C(CH₂OH)₃ HCl (BisTris⁺HCl). A small kinetic hydrogen-deuterium isotope effect ($\text{k}{}_{\mathrm{HS}}\text{k}{}_{\mathrm{DS}}\text{= 1.10}$) was observed for the general acid catalysed portion of the addition reaction. The kinetics of bisulfite elimination from 5-fluoro-5,6-dihydrouracil-6-sulfonate were studied in ethanolamine buffers. As previously observed with 1,3-dimethyl-5,6-dihydrouracil-6-sulfonate, this reaction is subject to general base catalysis and exhibits a large kinetic hydrogen-deuterium isotope effect (). The kinetic results for the addition reaction are consistent with a multistep reaction pathway involving the initial formation of an oxyanion sulfite addition intermediate (II) which subsequently adds a proton and undergoes tautomerization to yield the final 5-fluoro-5,6-dihydrouracil-6-sulfonate product. Thus the elimination of bisulfite from 5-fluoro-5,6-dihydrouracil-6-sulfonate probably proceeds by an ElcB mechanism which involves, at relatively low concentrations of general base, rate determining general base catalyzed proton abstraction from carbon 5 to yield intermediate II followed by the rapid elimination of sulfite to yield 5-fluorouracil. These results may be related to both the enzymatically catalyzed dehalogenation of bromoand iodouracil and the methylation of deoxyuridylate by thymidylate synthetase.     

Ruthenium(II) complexes derived from 2-(methylamino)pyridine

2-(Methylamino)pyridine reacts with RuCl₂(CO)₃ to give a carbamoyl complex, [$\text{Ru(C(O)N(CH}{}_3\text{)(C}{}_5\text{H}{}_4\text{N}$)Cl(CO)₂], which yields with pyridine (py) and acetylacetone (Hacac), respectively, [$\text{Ru(C(O)N(CH}{}_3\text{)C}{}_5\text{H}{}_4\text{N}$)Cl(CO)₂(py)] and [$\text{Ru(C(O)N(CH}{}_3\text{)C}{}_5\text{H}{}_4\text{N}$)(CO)₂(acac)]. These complexes are characterized spectroscopically. The amino group of the ligand is carbonylated and the resulted carbamoyl ligand is chelating through a pyridine ring-N and a carbamoyl-C atom. 2-Aminopyridine and 2-aminopyrimidine react similarly with RuCl₂(CO)₃ to give the corresponding carbamoyl complexes.     

Enhanced rates of acyl transfer to a neighboring hydroxyl group

2-Hydroxymethyl-4-nitrophenyl trimethylacetate is rapidly converted, by an intramolecular pathway, to its benzyl ester counterpart in aqueous solutions of dilute buffers. Intramolecular acyl migration is favored by a factor of 10⁵ over intermolecular transfer of the trimethylacetyl group to surrounding water molecules. The activation parameters of the reaction demonstrate that the rate acceleration is primarily entropic in origin. At constant pH, the apparent first-order rate constant for intramolecular acyl migration displays a linear dependence on the concentration of the basic component of the buffer. For catalysis by imidazole, a solvent deuterium isotope effect of $\text{k}{}_{\mathrm{<mtext>H</mtext>}}\text{k}{}_{\mathrm{<mtext>D</mtext>}}\text{= 2.4}$ is observed, in accord with a general base-catalyzed pathway. Similarities between intramolecular and intracomplex transacylations are discussed with the conclusion that the migration of a trimethylacetyl group from the phenolic oxygen atom of a 2-hydroxymethyl-4-nitrophenol to the adjacent benzylic oxygen atom provides an accurate model for acylation of the serine hydroxyl group at the active site of α-chymotrypsin by nitrophenyl esters.     

Conversion of thyroxine to 3,3',5'-triiodothyronine (reverse-T3) by a soluble enzyme system of rat liver

Infrared spectra of N₂O in a variety of solvents and in the brain of a dog under typical conditions of halothane-N₂O anesthesia have been determined. The appearance or disappearance of N₂O in the brain was readily followed as N₂O was administered or withdrawn. The sites in brain were of two major types; one, with ν₃ = 2229.8 ± 0.4 cm^?1 and $\text{Δν}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 13.0 ± 0.6}\text{cm}{}^{\mathrm{?1}}$, is rather like the polar site in water and the other, with ν₃ = 2216.8 ± 0.8 cm^?1 and $\text{Δν}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 9.6 ± 1.0}\text{cm}{}^{\mathrm{?1}}$, is non-polar and is probably associated with membrane lipid. The significant variations in the antisymmetric stretch (ν₃) of N₂O as the polarity and other properties of the medium (solvent) vary make possible the characterization of $\text{in tissue}$ sites occupied by this anesthetic.     

Dipole moments of random coil polypeptides

The Rotational Isomeric States model is applied to calculate dipole moments of polypeptides of the twenty natural α-amino acids in the random coil state. Dipole moments of each repeat unit (μ_i), are evaluated using a quantum mechanics procedure. Dipole moment ratios ($\text{D}{}_{\mathrm{x}}\text{=}\text{〈μ}{}^2\text{〉}\text{x}\text{μ}{}_{\mathrm{i}}{}^2\text{,}\text{x = number of repeat units}$) of homopolypeptides are calculated and extrapolated to x →?. With a few exceptions, D_? = 0.36 ± 0.1. Ten actual proteins and three enzymes are also studied; their dipole ratios (D_x′ =〈μ〉/x) range from 7.34 to 10.57 in 10^?59 C² m² (6.6–9.5 D²). Diffferences in the values of D_x′ are due mainly to the different contributions, μ_i, of the amino acid residues contained in each polymer, whereas the sequence of amino acids has a very minor effect.     

Fluorine NMR studies of fluorocinnamoylchymotrypsins

Three monofluorocinnamoylchymotrypsins have been examined at pH 4 by fluorine NMR spectroscopy. Protein-induced fluorine chemical shifts are quite large (~7 ppm) when fluorine is present at the para position but nearly zero for ortho fluorine. The shifts roughly parallel those observed in complexes formed between the enzyme and the analogous N-acetylfluorophenylalanines, suggesting a similarity in molecular environment for the aromatic ring in both systems. Little correlation is found, however, between the shifts for the acylenzymes and those of the corresponding enzyme-cinnamate complexes, indicating that the environment for the aromatic ring in the complexes is dissimilar from that experienced by the aromatic group in the acylated enzyme. Solvent isotope effects ($\text{H}{}_2\text{O}\text{D}{}_2\text{O}$) on the fluorine chemical shifts for the fluorocinnamoylchymotrypsins are small and downfield. Fluorine NMR observations suggest that the presence of the fluorocinnamoyl group greatly stabilizes the enzyme toward denaturation in 8 m urea.     

A proposed mechanism for light emission by bacterial luciferase involving dissociative electron transfer

A new mechanism that involves dissociative electron transfer in the energy transducing step is set forward for bacterial luciferase catalyzed light emission. The proposal involves (1) dissociation of the 4a-hydroperoxyflavin to a flavin radical and ${}^{\mathrm{?}}\text{O}{}_2{}^{\mathrm{?}}$, accounting for 570 and 620nm absorption, (2) ${}^{\mathrm{?}}\text{O}{}_2{}^{\mathrm{?}}$ addition to the aldehyde carbonyl to form a peroxyl radical, (3) abstraction of H from an enzyme thiol group to form RCH(OOH)OH, (4) thiyl radical abstraction of the H on C in RCH(OOH)OH, a step which can show a $\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}$ of ca. 4, and (5) dissociative electron- transfer, a highly exothermic step that leads to a protonated flavin excited state, a carboxylic acid and water.