Mechanistic studies of sodium pump

Sodium pump was the first ion pump discovered. A member of the family of active transporters that catalyze adenosine 5′-triphosphate hydrolysis by forming a phosphorylated enzyme intermediate, sodium pump couples the energy released to unequal countertransport of sodium and potassium ions. The ion gradient generated by the pump is important for a variety of secondary physiological processes ranging from metabolite transport to electrical excitation of nerve and muscle. Selected experiments relating structure to function are reviewed.     

Interfacial catalysis by lipases

We designed a convenient, specific, sensitive and continuous lipase activity assay using natural long-chain triacylglycerols (TAGs). Oil was extracted from Parinari glaberrimum seed kernels and the purified TAGs used as a substrate for detecting low levels of lipase activities. The purified TAGs are naturally fluorescent. The presence of detergents above their critical micellar concentration dramatically increases the fluorescence of the parinaric acid released by various lipases. This increase is linear with time and proportional to the amount of lipase added. Quantities as low as 0.1 ng of pure pancreatic lipase could be detected under standard conditions (pH 8).

The interfacial activation of human pancreatic lipase (HPL) probably involves the motion of a lid covering the active site of the enzyme. We observed that the presence of either bile salts or a small proportion of water-miscible organic solvents (called activator compounds) considerably enhances the enzymatic activity of HPL on a monomeric solution of tripropionin. This finding suggests that the activator compounds may favor the opening of the lid. This hypothesis was checked by comparing the immunoreactivity of HPL and HPL with a mini-lid (HPL(-lid)) towards anti-HPL monoclonal antibodies (mAbs), in the presence and absence of the activator compounds.     

Design and synthesis of conformationally restricted inhibitors of active thrombin activatable fibrinolysis inhibitor (TAFIa)

A series of 4,5,6,7-tetrahydro-1H-benzimidazole-5-carboxylic acid and 5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-7-carboxylic acid derivatives designed as inhibitors of TAFIa has been prepared via a common hydrogenation–alkylation sequence starting from the appropriate benzimidazole and imidazopyridine system. We present a successful design strategy using a conformational restriction approach resulting in potent and selective inhibitors of TAFIa. The X-ray structure of compound 5 in complex with a H333Y/H335Q double mutant TAFI indicate that the conformational restriction is responsible for the observed potency increase.     

Structural biology of human cannabinoid receptor-2 helix 6 in membrane-mimetic environments

We detail the structure and dynamics of a synthetic peptide corresponding to transmembrane helix 6 (TMH6) of human cannabinoid receptor-2 (hCB2) in biomembrane-mimetic environments. The peptide’s NMR structural biology is characterized by two α-helical domains bridged by a flexible, nonhelical hinge region containing a highly-conserved CWFP motif with an environmentally sensitive, Pro-based conformational switch. Buried within the peptide’s flexible region, W²⁵⁸ may hydrogen-bond with L²⁵⁵ to help stabilize the Pro-kinked hCB2 TMH6 structure and position C²⁵⁷ advantageously for interaction with agonist ligands. These characteristics of hCB2 TMH6 are potential structural features of ligand-induced hCB2 activation in vivo.     

Enzyme conformational alterations detected by partition column chromatography

In this paper, we demonstrate the ability of liquid-liquid partition chromatography (LLPC) to detect conformational alterations occurring in well-characterized enzymes. The conformational changes induced in dehydrogenases such as alcohol dehydrogenase (ADH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenases (LDH) and malate dehydrogenase (MDH) upon binding of ligand(s) were detectable by LLPC. The ligand-dependent equilibrium between two forms of citrate synthase (CS), glutamate-oxaloacetate transaminase (GOT), hexokinase (HK) and 3-phosphoglycerate kinase (PGK) could also be demonstrated. Furthermore, different conformational forms of some of the apoenzymes could also be detected and separated by LLPC. The results obtained here are discussed in relation to those obtained by other methods.     

Binding Thermodynamics of the Transition State Analogue Coformycin and of the Ground State Analogue 1-Deazaadenosine to Bovine Adenosine Deaminase

Binding of the transition state analogue coformycin and the ground state analogue 1-deaazadenosine to bovine adenosine deaminase have been thermody-namically characterized. The heat capacity changes for coformycin and 1-deazaadenosine binding are - 4.7 × 0.8 kJ/mole-K and -1.2 × 0.1 kJ/mole-K, respectively. Since the predominant source of heat capacity change in enzyme interactions are changes in the extent of exposure of nonpolar amino acid side chains to the aqueous environment and the hydrophobic effect is the predominant factor in native structure stabilization, we propose that the binding of either class of ligand is associated with a stabilizing enzyme conformational change with coformycin producing the far greater effect Analysis of the T dependence of the second order rate constant for formation of the enzyme/coformycin complex further reveals that the conformational change is not rate limiting. We propose that the enzyme may facilitate catalysis via the formation of a stabilizing conformation at the reaction transition state.     

Conformational investigation of a novel, dipeptide based molecular scaffold

Summary The conformational features of a novel, dipeptide-based molecular scaffold are described. Four model systems of a trisubstituted 1,4-diazepine-3-one system, varying in the chirality and amino acid within the ring system, have been investigated by high-resolution NMR and metric-matrix distance geometry calculations. Because of the small number of protons within the scaffold, nuclear Overhauser effects provide only limited conformational information. Instead, extensive use of scalar¹H−H¹ and¹H−¹³C coupling constants was utilized in the refinement. The resulting conformations of the model systems provide insigh into the expected topological orientation of the amino acids or chemical functionalities and attached to the seven-membered ring system, the first step of the utilization of this scaffold in the rational design of peptidomimetics.     

Conformationally Restricted Chiral Peptide Nucleic Acids Derived from Azetidines

Abstract

The initial experiments towards the chemical synthesis of conformationally rigid peptide nucleic acid analogues with azetidine moieties have been described.     

Three-dimensional chromatin ensemble reconstruction via stochastic embedding

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