Enzyme catalysis from improved packing in their transition-state structures.

The binding of ligands to proteins can be enhanced through improved packing within the proteins that may, or may not, occur with conformational change. Enzymes can similarly improve their catalytic magic through better packing in the transition state (TS) for reaction. In principle, the improved packing demands no more than the minute shortening of non-covalent interactions throughout much of the structure of the protein (positively cooperative binding).

Ligand binding energy and enzyme efficiency from reductions in protein dynamics.

Tetrameric rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (GAPDH; EC 1.2.1.

Understanding noncovalent interactions: ligand binding energy and catalytic efficiency from ligand-induced reductions in motion within receptors and enzymes.

Noncovalent interactions are sometimes treated as additive and this enables useful average binding energies for common interactions in aqueous solution to be derived. However, the additive approach is often not applicable, since noncovalent interactions are often either mutually reinforcing (positively cooperative) or mutually weakening (negatively cooperative). Ligand binding energy is derived (positively cooperative binding) when a ligand reduces motion within a receptor.

The N-linked oligosaccharides of aminopeptidase N from Manduca sexta: site localization and identification of novel N-glycan structures.

Mass spectrometric studies on the N-linked glycans of aminopeptidase 1 from Manduca sexta have revealed unusual structures not previously observed on any insect glycoprotein. Structure elucidation of these oligosaccharides was carried out by high-energy collision-induced dissociation (CID) using a matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) tandem mass spectrometer. These key experiments revealed that three out of the four N-linked glycosylation sites in this protein (Asn295, Asn623 and Asn752) are occupied with highly fucosylated N-glycans that possess unusual difucosylated cores.

Importance of structural tightening, as opposed to partially bound States, in the determination of chemical shift changes at noncovalently bonded interfaces.

Two models (A and B) have been proposed to account for decreased downfield chemical shifts of a proton bound by noncovalent interactions at a ligand/antibiotic interface as the number of ligand/antibiotic interactions is decreased. In model A, the proton involved in the noncovalent bond suffers a smaller downfield shift because the bond is, with a relatively large probability, broken, and not because it is longer. In model B, the proton involved in the noncovalent bond suffers a smaller downfield shift because the bond is longer, and not because it is, with a relatively large probability, broken.

Order changes within receptor systems upon ligand binding: receptor tightening/oligomerisation and the interpretation of binding parameters.

Recent hydrogen-deuterium exchange experiments have highlighted tightening and loosening of protein structures upon ligand binding, with changes in bonding (DeltaH) and order (DeltaS) which contribute to the overall thermodynamics of ligand binding. Tightening and loosening show that ligand binding respectively stabilises or destabilises the internal structure of the protein, i.e.

Fragmentation characteristics of neutral N-linked glycans using a MALDI-TOF/TOF tandem mass spectrometer.

The fragmentation characteristics of native and permethylated oligosaccharides using a matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight tandem mass spectrometer are described. The use of two MALDI matrixes, alpha-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), is shown to control the nature and extent of fragmentation observed in collision-induced dissociation experiments on synthetic oligosaccharides. CHCA promotes the occurrence of glycosidic cleavages, whereas DHB promotes a wide range of fragmentations.

Noncovalent interactions: defining cooperativity. Ligand binding aided by reduced dynamic behavior of receptors. Binding of bacterial cell wall analogues to ristocetin A.

Changes in the relative populations of the monomer and asymmetric dimer forms of ristocetin A, upon binding of two molecules of ligand, suggest that ligand binding is negatively cooperative with respect to dimerization. However, strong hydrogen bonds formed in the binding sites of the ligands are reinforced in the dimer relative to the monomer, and the barrier to dissociation of the dimer is increased upon binding of the ligands. It is concluded that the interactions which are common in the binding of both ligands are made with positive cooperativity with respect to those involved in dimerization.

How can enzymes be so efficient?

Dudley Williams and his colleagues discuss how ligands can gain binding energy to their receptors, and substrate transition states to their enzymes, by tightening the protein structures, with a decrease in their dynamic behaviour.

Kinetic barriers and ordering of non-covalently bound states.

Binding of a dimer of a glycopeptide antibiotic to two molecules of a ligand that are bound to a membrane surface (by a hydrocarbon anchor) has been investigated. This binding on a surface is cooperatively enhanced (surface enhancement) relative to the binding in solution, because the former occurs intramolecularly on a template. Previously a correlation between surface enhancement and thermodynamic stability of the dimer in free solution (Kdimsol) was hypothesised.

Ligand binding energy and catalytic efficiency from improved packing within receptors and enzymes.

Some small molecules bind to their receptors, and transition states to enzymes, so strongly as to defy current understanding. We show that in the binding of biotin to streptavidin, the streptavidin structure becomes better packed. We conclude that this contraction of the streptavidin structure promotes biotin binding.

Binding of an inhibitor of the p53/MDM2 interaction to MDM2.

The mode of action of the secondary metabolite chlorofusin, which antagonises the interaction between p53 and MDM2, involves direct binding to the N-terminal domain of MDM2.

Changes in motion vs. bonding in positively vs. negatively cooperative interactions.

From a consideration of the interactions between non-covalent bonds, it is concluded that positively cooperative binding will occur with a benefit in enthalpy and a cost in entropy, and that negatively cooperative binding will occur with a cost in enthalpy and a benefit in entropy; experimental data support these conclusions.

Cooperative binding interactions of glycopeptide antibiotics.

Glycopeptide antibiotics of the vancomycin group bind to bacterial cell wall analogue precursors, and typically also form dimers. We have studied the interplay between these two sets of noncovalent bonds formed at separate interfaces. Indole-2-carboxylic acid (L) forms a set of hydrogen bonds to the glycopeptide antibiotic chloroeremomycin (CE) that are analogous to those formed by N-Ac-D-Ala.

Mechanism of the regulation of type IB phosphoinositide 3OH-kinase byG-protein betagamma subunits.

Type IB phosphoinositide 3OH-kinase (PI3K) is activated by G-protein betagamma subunits (Gbetagammas). The enzyme is soluble and largely cytosolic in vivo. Its substrate, PtdIns(4,5)P(2), and the Gbetagammas are localized at the plasma membrane.

The Vancomycin Group of Antibiotics and the Fight against Resistant Bacteria.

A last line of defence against "superbugs" are the vancomycin group antibiotics. This review describes the determination of their mode of action, and a mechanism of resistance to them. Remarkably, this mechanism of resistance can be overcome without directly modifying the binding site of the antibiotics for the cell-wall precursors of pathogenic bacteria.