Jeremy R. Knowles

[8] Photoaffinity labeling

Publisher Summary This chapter discusses the photoaffinity labeling which could be used as a method that allows to unleash the reagent at a particular time and place, when the chemical affinity labeling restricts it. The chapter notes that the possibility that a labile group of appropriate reactivity cannot be incorporated into the ligand molecule without excessive disturbance of the recognition process, there are two limitations to the affinity labeling approach. The first challenge; the range of chemical reactivity of groups that can be incorporated into the ligand is limited by the fact that these groups must not react so rapidly with water that they are destroyed hydrolytically before the ligand that carries them can reach the binding site. And secondly, it is becoming clear that some biological problems require a reagent whose reactivity remains masked until the experimenter chooses to activate it. Both of the two limitations of classical chemical affinity labeling discussed above can in principle be circumvented by the use of a photogenerated reagent.

Reduction of aryl azides by thiols: Implications for the use of photoaffinity reagents

Abstract Aryl azides are rapidly reduced by dithiothreitol at room temperature to the corresponding aryl amines. Glutathione and 2-mercaptoethanol react much more slowly. The relevance of this reaction to experiments involving aryl azide photoaffinity reagents is discussed.

[16] Chiral [16O, 17O, 18O] phosphoric monoesters as stereochemical probes of phosphotransferases

Publisher Summary This chapter considers the mechanistic value of determining the stereochemical consequence of an enzyme-catalyzed phosphoryl-transfer reaction and focuses on the methods for the synthesis and stereochemical analysis of chiral [ 18 O, 17 O, 18 O] phosphoric monoesters. First chapter discusses the synthesis of [1(R)- 16 O, 17 O, 18 O] phospho-(S)-propane-1,2-dioP by the ephedrine route. P 17 OCl 3 , prepared from H 2 17 O and PCl 5 in 85% yield, is allowed to react with (–)-ephedrine to yield the two diastereoisomeric chloro adducts in the approximate ratio 9: 1. Other method allows the stereospecific replacement of peripheral oxygen of a phosphoric diester either by sulfur or by oxygen. It is reported that the success of any stereospecific synthesis of chiral phosphoric esters is measured by its ability to yield a variety of esters of high isotopic purity that have a known configuration at phosphorus. To determine the absolute configuration of a labeled phosphoric monoester, one needs to define the absolute R or S sense of the peripheral oxygen isotopes of the phosphoryl group. The sense of isotopic substitution is determined in a diastereoisomer with respect to a separate chiral carbon center of known absolute configuration.

[18] Synthesis of ribulose 1,5-bisphosphate: Routes from glucose 6-phosphate (via 6-phosphogluconate) and from adenosine monophosphate (via ribose 5-phosphate)

Publisher Summary This chapter describes the synthesis of ribulose 1,5-bisphosphate. The synthesis of ribulose 1,5-bisphosphate (RuBP) is accomplished by two routes: (1) glucose 6-phosphate and (2) adenosine monophosphate. These routes provide procedures suitable for preparing several hundred grams of RuBP and of the major intermediates lying between starting materials and this metabolite. The enzymes required are all used in an immobilized form. Both routes require ATP cofactor recycling and the procedure includes the details of an improved synthesis of the acetyl phosphate used as the ultimate phosphate donor for this recycling. The route based on glucose 6-phosphate (G-6-P) also illustrates a convenient procedure for the regeneration of nicotinamide adenine dinucleotide phosphate [NAD(P)] from nicotinamide adenine dinucleotide phosphate dehydrogenase [NAD(P)H] under anaerobic conditions, based on the conversion of α-ketoglutarate to glutamate. It is important to minimize the exposure of the RuBP to isomerizing conditions during and after preparation. The procedures for the preparation of RuBP are also summarized in the chapter. Glucose 6-phosphate is prepared using hexokinase-catalyzed phosphorylation and adenosine triphospahte (ATP) regeneration. Two methods employed for RuBP determination are the coupled-enzyme and radiometric method.

Synthesis and evaluation of two new inhibitors of EPSP synthase

Abstract The enzyme EPSP synthase, EPSPS, (EC 2.5.1.19) catalyzes an unusual transfer reaction of the enolpyruvoyl moiety from phosphoenol pyruvate ( 2 , PEP) regiospecifically to the 5-OH of shikimate 3-phosphate ( 1 , S3P) to form 5-enol-pyruvoylshikimate 3-phosphate ( 3 , EPSP). Two new inhibitors, ( 4 , and 5 ) were prepared to probe the S3P binding site.

Retention of the oxygens at C-2 and C-3 of D-ribulose 1,5-bisphosphate in the reaction catalyzed by ribulose-1,5-bisphosphate carboxylase.

Ribulose-1,5-bisphosphate carboxylase catalyzes the conversion of D ribulose 1,5-bisphosphate and CO2 to 3-phospho-D-glycerate, with retention of the oxygen atoms at both C-2 and C-3 of the substrate. This observation is consistent with mechanistic pathways involving an enediol intermediate and eliminates suggested mechanisms that involve covalent intermediates between the enzyme and ribulose 1,5-bisphosphate in which the substrate oxygen at C-2 or C-3 is compulsorily lost.

Chapter 25. Bacterial Resistance to β-Lactams: The β-Lactamases

Publisher Summary The β-lactamases are monomeric enzymes typically of molecular weight of around 30,000 that catalyze the hydrolysis of the β-lactam ring of a variety of penam (I) and cephem (II) derivatives. β-Lactamases have been purified from a number of sources. The most important sources are the enzymes from Staphylococcus aureus, Bacillus cereus, Bacillus licheniformis , and Escherichia coli . These enzymes are discussed of in the chapter. β-lactamases are extremely efficient in catalyzing the hydrolysis of their more susceptible substrates. Bacteria produce β-lactamases to resist the lethal consequences of the exposure to β-lactam antibiotics. It rapidly hydrolyzes the β-lactam to the 8-amino acid derivative. These enzymes are extremely efficient catalysts, they may be constitutive or inducible, can be coded for chromosomally or by readily transferable plasmids, and they may be largely intracellular, extracellular, or, for gram-negative bacteria, predominantly periplasmic. While it is clear that the permeability of the cells membrane to the antibiotic sharply affects β-lactam potency, it appears that most commonly encountered resistance to β-lactams derives from the presence of the β-lactamase. Although the central role of the β-lactamases in resistance is clear, efforts to overcome this resistance by using β-lactamase inhibitors have not proved to be very fruitful, because the specificity of β-lactamases is wide and broad-spectrum β-lactamase inhibitors are not available. This situation is changing, and the tactical inclusion of β-lactamase-inhibitory function into an antibiotic or the use of a β-lactamase inhibitor in synergy with a susceptible but effective antibiotic is becoming attractive. This chapter reviews the properties of some of the better studied β-lactamases and the interaction with E.coli RTEM β-lactamase of a number of recently discovered β-lactams containing unusual structural features.

The preparation and photolysis of 3-aryl-3H-diazirines

The preparation of a number of 3-aryl-3H-diazirines is reported. On irradiation, these materials undergo both photolytic fragmentation to the arylcarbene and photoisomerisation to the linear diazo-compound which is then itself photolysed. The existence of a second intermediate is also apparent from the spectral changes observed.

Energetics of enzyme catalysis. I. Isotopic experiments, enzyme interconversion, and oversaturation.

An enzyme-catalyzed interconversion of one substrate, S, and one product P, by an enzyme that exists in two forms E1 and E2 where E1 binds S and E2 binds P, is considered S + E1 in equilibrium E1S in equilibrium E2P in equilibrium E2 + P. Under reversible conditions (where the concentrations of S and P are not far removed from their equilibrium values) it is shown that, in addition to the usual unsaturated and saturated behaviour there exists a third regime at high substrate concentration: the oversaturated region. In this region, the rate-limiting transition state is the interconversion of the unliganded forms of the enzyme: E1 and E2. Expressions for six different experiments involving deuterium, tritium and 14C labels are presented. By considering the results from these experiments, the nature and importance of the enzyme interconversion steps can be elucidated.

Energetics of enzyme catalysis. II. Oversaturation, case diagrams, reversible and irreversible behaviour

Most enzymes react in vivo under reversible conditions where the substrate and product concentrations are not far removed from equilibrium values. Under these conditions when the concentration of substrate is increased, in addition to the usual unsaturated and saturated behaviour we find a third type of kinetic regime at high substrate concentration-oversaturation. In this regime the rate limiting transition state involves interconversion of free enzyme forms. For a one substrate/one product enzyme, case diagrams can be constructed which depict the kinetic behaviour as a function of substrate and product concentrations. Six different cases are found and are discussed with the relevant free energy profiles. A systematic procedure is described for the investigation and construction of the case diagram.