R. S. Chittock

Hydrogen bonding and protein perturbation in beta-lactam acyl-enzymes of Streptococcus pneumoniae penicillin-binding protein PBP2x.

A soluble form of Streptococcus pneumoniae PBP2x, a molecular target of penicillin and cephalosporin antibiotics, has been expressed and purified. IR difference spectra of PBP2x acylated with benzylpenicillin, cloxacillin, cephalothin and ceftriaxone have been measured. The difference spectra show two main features. The ester carbonyl vibration of the acyl-enzyme is ascribed to a small band between 1710 and 1720 cm-1, whereas a much larger band at approx. 1640 cm-1 is ascribed to a perturbation in the structure of the enzyme, which occurs on acylation. The protein perturbation has been interpreted as occurring in beta-sheet. The acyl-enzyme formed with benzylpenicillin shows the lowest ester carbonyl vibration frequency, which is interpreted to mean that the carbonyl oxygen is the most strongly hydrogen-bonded in the oxyanion hole of the antibiotics studied. The semi-synthetic penicillin cloxacillin is apparently less well organized in the active site and shows two partially overlapping ester carbonyl bands. The penicillin acyl-enzyme has been shown to deacylate more slowly than that formed with cloxacillin. This demonstrates that the natural benzylpenicillin forms a more optimized and better-bonded acyl-enzyme and that this in turn leads to the stabilization of the acyl-enzyme required for effective action in the inhibition of PBP2x. The energetics of hydrogen bonding in the several acyl-enzymes is discussed and comparison is made with carbonyl absorption frequencies of model ethyl esters in a range of organic solvents. A comparison of hydrolytic deacylation with hydroxaminolysis for both chymotryspin and PBP2x leads to the conclusion that deacylation is uncatalysed.

A critical analysis of the use of radiation inactivation to measure the mass of protein.

Measurements are presented of the radiation inactivation of four enzymes exposed to a 6 MeV proton beam. It has long been thought that the measurement of the susceptibility of an enzyme to ionizing radiation can be used to determine its molecular mass. Results are frequently interpreted using the empirical analysis of Kempner and Macey (Biochim. Biophys. Acta 163, 188-203, 1963). We examine this analysis and discuss the validity and limitations of the assumptions on which it is based. Our results indicate that the specific biochemical properties of each enzyme make a significant contribution to its radiation sensitivity.

The quantum yield of luciferase is dependent on ATP and enzyme concentrations

Abstract The Quantum Yield of firefly luciferase relative to its substrate luciferin is a function of enzyme and ATP concentrations. The implications for molecular electronic applications are discussed.

Modulation of firefly luciferase bioluminescence at bioelectrochemical interfaces.

This paper describes a method by which the activity of an immobilized enzyme can be modulated electrochemically at an electrode. The particular example studied, involving the enzyme firefly luciferase being immobilized in a gelatin film of thickness <1 μm, provides a useful model system since changes in the catalytic activity can be measured instantaneously through the natural bioluminescent emission. Using this biointerfacial arrangement, we have been able to demonstrate the reversible switching off and on of the enzymes activity. Through a series of mechanistic studies, we have been able to determine that the bioluminescence response is modulated (on long time scales) as a consequence of the electrochemical depletion of protons at the electrode interface resulting in a local increase in pH.

Optical Switching and Amplification Based on the Enzyme Luciferase

Abstract Films containing luciferase and its substrates were made to act as optical memories and correlators.

Micrometre-scale bioluminescent enzyme photopatterning for bioelectronics applications

Abstract The enzyme firefly luciferase was patterned onto gold and glass surfaces at micrometre resolution using a novel photopatterning technique. The bioluminescence emitted on addition of the enzyme substrates luciferin and ATP allows these patterns to be visualised. Electrical switching of enzyme activity has been achieved by using enzyme immobilised on a gold surface as one electrode in an electrolysis cell. These techniques have applications in biosensor construction and in bioelectronics.

Infrared Spectroscopy of Interactions Between Antibiotics and β-Lactamases/Transpeptidases

The serine proteinase enzymes form a disparate family that encompasses the digestive enzymes chymotrypsin and trypsin, thrombin, which is involved in blood clotting, as well as bacterial enzymes such as subtilisin. The structures of the mammalian enzymes fall into one class and are composed predominantly of β-sheet, while the bacterial enzymes are predominantly α-helical. Despite this difference in structure all serine proteinases have an essentially identical catalytic apparatus. This is comprised of the relay system [Asp-His-Ser], the oxyanion hole and a specificity conferring sidechain binding pocket. The serine of the relay system acts as a nucleophile which attacks the substrate at the susceptible carbonyl group, where the peptide chain is cleaved. The His residue, strongly hydrogen bonded to the Ser acts as a general base to remove a proton from the Ser in both steps of the reaction (see below) and to provide a proton, while acting as an acid, for the leaving nitrogen atom in acylation or the serine in deacylation. The Asp residue serves to stabilise the conformation of the His residue as required for its action as a general base. The oxyanion hole is comprised of two backbone amide nitrogens, whose hydrogens are so placed to hydrogen bond to the susceptible carbonyl that is to be attacked by the serine residue. The predefined spatial organisation of these amide hydrogens ensures that there is no entropy requirement upon substrate or acyl group interaction. The oxyanion hole has been supposed to stabilise the negative charge which develops in the transition states for each of the two steps of the enzyme-catalysed reaction:where EA is the acylenzyme. The C-terminal part of the substrate is lost in the acylation step, characterised by k2, while the N-terminal part is lost as a consequence of deacylation, characterised by k3. The acylation step generates a unique ester bond in the structure of the covalently bonded enzyme substrate complex. The deacylation step represents a hydrolysis of the ester intermediate. The acylation and deacylation steps are almost mirror images of one another. The amide bonds of proteins or peptides are hydrolytically resistant and a complex mechanism has evolved to cope with this. The unique ester group of the acylenzyme is amenable to study using IR spectroscopy since it absorbs at a frequency that is higher than the peptide amide groups, although 13C=Oisotope labelling is required to ensure band assignment, since perturbation of the protein spectrum by formation of the acylenzyme can, at low pH, interfere with the ester band(s) [1–5].

Electrochemical modulation of bioluminescence

The electrochemical modulation of the bioluminescence of firefly luciferase is described, showing that the enzyme can be switched ON and OFF, as a function of both the magnitude and duration of the applied potential.