Vernon E. Anderson

Experimental and computational analysis of the transition state for ribonuclease A-catalyzed RNA 2′-O-transphosphorylation

Enzymes function by stabilizing reaction transition states; therefore, comparison of the transition states of enzymatic and nonenzymatic model reactions can provide insight into biological catalysis. Catalysis of RNA 2′-O-transphosphorylation by ribonuclease A is proposed to involve electrostatic stabilization and acid/base catalysis, although the structure of the rate-limiting transition state is uncertain. Here, we describe coordinated kinetic isotope effect (KIE) analyses, molecular dynamics simulations, and quantum mechanical calculations to model the transition state and mechanism of RNase A. Comparison of the 18O KIEs on the 2′O nucleophile, 5′O leaving group, and nonbridging phosphoryl oxygens for RNase A to values observed for hydronium- or hydroxide-catalyzed reactions indicate a late anionic transition state. Molecular dynamics simulations using an anionic phosphorane transition state mimic suggest that H-bonding by protonated His12 and Lys41 stabilizes the transition state by neutralizing the negative charge on the nonbridging phosphoryl oxygens. Quantum mechanical calculations consistent with the experimental KIEs indicate that expulsion of the 5′O remains an integral feature of the rate-limiting step both on and off the enzyme. Electrostatic interactions with positively charged amino acid site chains (His12/Lys41), together with proton transfer from His119, render departure of the 5′O less advanced compared with the solution reaction and stabilize charge buildup in the transition state. The ability to obtain a chemically detailed description of 2′-O-transphosphorylation transition states provides an opportunity to advance our understanding of biological catalysis significantly by determining how the catalytic modes and active site environments of phosphoryl transferases influence transition state structure.

Structure of Hexadienoyl-CoA Bound to Enoyl-CoA Hydratase Determined by Transferred Nuclear Overhauser Effect Measurements: Mechanistic Predictions Based on the X-ray Structure of 4-(Chlorobenzoyl)-CoA Dehalogenase†

The structure of the substrate analog 2,4-hexadienoyl-coenzyme A (HD-CoA) bound to the enzyme enoyl-CoA hydratase has been determined using transferred nuclear Overhauser enhancement (TRNOE) spectroscopy. NOEs between the adenine H8 proton and several pantetheine protons in the bound form of HD-CoA indicate that the overall structure of the CoA molecule is bent, while NOEs between adenine and ribose protons indicate that the conformation about the glycosidic bond is anti. The absence of long range NOEs along the pantetheine moiety is consistent with this region of the molecule being bound in an extended conformation. In addition, NOEs between the vinylic protons indicate that the HD moiety is s-trans about C3-C4. The conformation of the CoA portion of bound HD-CoA is strikingly similar to that of the CoA portion of 4-(hydroxybenzoyl)-CoA bound to the active site of 4-(chlorobenzoyl)-CoA dehalogenase [Benning, M. M., et al. (1996) Biochemistry 35, 8103-8109]. The structural similarity of the ligands along with the primary sequence homology validates the modeling of the enoyl-CoA hydratase structure with the 4-(chlorobenzoyl)-CoA dehalogenase backbone. The homology modeling allows the prediction that the enoyl-CoA substrates are bound in an s-cis conformation about C1-C2 and that Glu 144 is present at the active site and can function as a general acid/base.

Detection of acyl-coenzyme a thioester intermediates of fatty acid β-oxidation as the N-acylglycines by negative-ion chemical ionization gas chromatography—Mass spectrometry☆

An analytical method for the separation and quantitation of acyl-CoA thioesters by gas chromatography-mass spectrometry is described. The method utilizes glycine aminolysis of the acyl-CoA thiolesters, esterification with pentafluorobenzyl bromide followed by gas chromatographic separation, and detection by negative chemical ionization mass spectrometry of the N-acylpentafluorobenzyl glycinates. The glycine aminolysis provides over 100-fold discrimination against oxygen esters and obviates the difficulty of removing trace contaminants of free fatty acids. The limit of detection of the described methodology for palmitoyl-CoA has been found to be 300 fmol, which improves at shorter chain lengths. Baseline separation was obtained for a standard mixture of seven acyl-CoAs (60 pmol injected) containing butyryl-CoA, hexanoyl-CoA, octanoyl-CoA, decanoyl-CoA, lauroyl-CoA, myristoyl-CoA, and palmitoyl-CoA. The above procedure is also applicable to the alpha-beta unsaturated and 3-hydroxyacyl-CoA derivatives, making it possible to quantify all of the intermediates in fatty acid oxidation, except the 3-ketoacyl-CoAs, in a single procedure.

3-Hydroxy-3-methylglutaryldithio-CoA: utility of an alternative substrate in elucidation of a role for HMG-CoA lyase's cation activator

(S)-(3-Hydroxy-3-methyl-1-thionoglutaryl)-Coenzyme A (HMG[= S]CoA), a dithioester analog of (S)-(3-hydroxy-3-methylglutaryl)-CoA (HMG-CoA), acts as an efficient alternative substrate for avian HMG-CoA lyase. Detection of product formation by HPLC, UV absorbance and coupled enzyme assays indicates that HMG[= S]CoA cleavage yields acetyl[= S]CoA and acetoacetate. HMG[= S]CoA binds to the lyase with a Km of 13 microM and undergoes the cleavage reaction at a maximal rate which is 20% of that observed with HMG-CoA. The enzyme-catalyzed cleavage of both HMG-CoA and HMG[= S]CoA is stimulated by the divalent cations Mg2+ and Mn2+. Mg2+ produces a 2-fold higher stimulation of HMG-CoA cleavage than that observed with Mn2+. In contrast, stimulation of HMG[= S]CoA cleavage is nearly seven times higher with Mn2+ than with Mg2+. Not only is the stimulation of enzymatic activity dependent on the cation, but also the Km values for Mg2+ and Mn2+ are dependent upon the substrate used. In contrast, the Km values for HMG-CoA and HMG[= S]CoA are not markedly dependent on the identity of the divalent cation. These results are compatible with the initial formation of a binary enzyme-substrate complex prior to binding of the divalent cation to produce a catalytically active enzyme-substrate-metal ternary complex.

A quantitative Raman spectroscopic signal for metal-phosphodiester interactions in solution.

Accurate identification and quantification of metal ion-phosphodiester interactions are essential for understanding the role of metal ions as determinants of three-dimensional folding of large RNAs and as cofactors in the active sites of both RNA and protein phosphodiesterases. Accomplishing this goal is difficult due to the dynamic and complex mixture of direct and indirect interactions formed with nucleic acids and other phosphodiesters in solution. To address this issue, Raman spectroscopy has been used to measure changes in bond vibrational energies due to metal interactions. However, the contributions of inner-sphere, H-bonding, and electrostatic interactions to the Raman spectrum of phosphoryl oxygens have not been analyzed quantitatively. Here, we report that all three forms of metal ion interaction result in attenuation of the Raman signal for the symmetric vibration of the nonbridging phosphate oxygens (nu(s)PO(2)(-)), while only inner-sphere coordination gives rise to an apparent shift of nu(s)PO(2)(-) to higher wavenumbers (nu(s)PO(2)(-)M) in solution. Formation of nu(s)PO(2)(-)M is shown to be both dependent on metal ion identity and an accurate measure of site-specific metal ion binding. In addition, the spectroscopic parameter reflecting the energetic difference between nu(s)PO(2)(-) and nu(s)PO(2)(-)M (DeltanuM) is largely insensitive to changes in phosphodiester structure but strongly dependent on the absolute electronegativity and hardness of the interacting metal ion. Together, these studies provide strong experimental support for the use of nu(s)PO(2)(-)M and DeltanuM as general spectroscopic features for the quantitative analysis of metal binding affinity and the identification of metal ions associated with phosphodiesters in solution.

Coenzyme A dithioesters: synthesis, characterization and reaction with citrate synthase and acetyl-CoA:choline O-acetyltransferase

Acyl dithioesters of CoA have been synthesized by transesterification. The alpha-hydrogens have a spectrally determined pKa of 12.5 +/- 0.14. The hydroxide catalyzed enolization rate is estimated to be 600 M-1.s-1. The absorbance of the dithioester, lambda max = 306 nm, can be used to monitor both the condensation and transesterification reactions that use CoA-Ac as a substrate. For citrate synthase at pH 7.4 Vmax = (4.0 +/- 0.4).10(-4) s-1 and Km = 53 +/- 7.5 microM, which are 2.10(-6) and 3.3-times the Vmax and Km values observed for CoAS-Ac, while for Ac-CoA: choline O-acetyltransferase (EC 2.3.1.6) at pH 7.0 Vmax = (1.1 +/- 0.2).10(-2) mumol.s-1.(mg protein)-1 and Km = 83 +/- 33 microM, which are 0.077 and 10-times the values observed with CoAS-Ac, respectively. The CoA dithioesters are stable at low pH, but hydrolyze with a second-order rate constant of 8.2.10(-2) M-1.s-1 at pH 11.4. The spectral properties of these dithioesters should allow these analogs to be used as probes of the structure of enzyme bound intermediates.

3-Hydroxy-3-methylglutaryldithio-coenzyme A: A potent inhibitor of Pseudomonas mevalonii HMG-CoA reductase

3-Hydroxy-3-methyl-1-thionoglutaryl-coenzyme A, a dithioester analog of 3-hydroxy-3-methylglutaryl-CoA, has been enzymatically synthesized using the HMG-CoA synthase catalyzed condensation of acetyl-CoA with 3-oxo-1-thionobutyryl-CoA. HMGdithio-CoA is a potent inhibitor of Pseudomonas mevalonii HMG-CoA reductase. Inhibition was mainly competitive with respect to HMG-CoA with a Kis of 0.086 +/- .01 microM and noncompetitive with respect to NADH with a Kis of 3.7 +/- 1.5 microM and a Kii of 0.65 +/- .05 microM in the presence of 110 microM (R.S)-HMG-CoA.