Deaminating enzyme labels for potentiometric enzyme immunoassay

Adenosine deaminase, asparaginase, and urease are examined as possible enzyme labels for immunoassays using potentiometric detection with the ammonia gas-sensing membrane electrode. Considerations of binding ability, retained activity, and stability reveal asparaginase to be the most effective enzyme label for immunoassay purposes. The utility of the potentiometric approach with this enzyme label is demonstrated via model hapten assays for dinitrophenyl groups and for cortisol.     

Regulation of enzyme activity in adsorptive enzyme systems

The kinetic behaviour of adsorptive enzyme systems with free and adsorbed enzyme forms in rapid equilibrium has been analysed. It has been shown that the dependences of enzymic reaction rate on substrate or “adsorptive effector” concentrations reveal the deviations from simple kinetic laws of Michaelis-Menten type (positive or negative kinetic co-operativity). Such kinetic anomalies should be observed when adsorption of the enzyme results in the changing catalytic properties and when the state of the equilibrium between free and bound enzyme forms depends on the presence of low molecular substances (substrates, coenzymes and various cellular metabolites). The physiological significance of adsorption-desorption processes for the enzyme activity regulation has been emphasized.     

Analysis of slow-binding enzyme inhibitors at elevated enzyme concentrations

The improvement in the characterization of slow-binding inhibitors achieved by performing experiments at elevated enzyme concentrations is presented. In particular, the characterization of slow-binding inhibitors conforming to a two-step mode of inhibition with a steady-state dissociation constant that is much lower than the initial dissociation constant with enzyme is discussed. For these systems, inhibition is rapid and low steady-state product concentrations are produced at saturating inhibitor concentrations. By working at elevated enzyme concentrations, improved signal-to-noise ratios are achieved and data may be collected at saturating inhibitor levels. Numerical simulations confirmed that improved parameter estimates are obtained and useful data to discern the mechanism of slow-binding inhibition are produced by working at elevated enzyme concentrations. The saturation kinetics that were unobservable in two previous studies of an enzyme inhibitor system were measured by performing experiments at an elevated enzyme concentration. These results indicate that consideration of the quality of the data acquired using a particular assay is an important factor when selecting the enzyme concentration at which to perform experiments used to characterize the class of enzyme inhibitors examined herein.     

A counterintuitive approach to treat enzyme deficiencies: use of enzyme inhibitors for restoring mutant enzyme activity

Pharmacological chaperone therapy is an emerging counterintuitive approach to treat protein deficiencies resulting from mutations causing misfolded protein conformations. Active-site-specific chaperones (ASSCs) are enzyme active-site directed small molecule pharmacological chaperones that act as a folding template to assist protein folding of mutant proteins in the endoplasmic reticulum (ER). As a result, excessive degradation of mutant proteins in the ER-associated degradation (ERAD) machinery can be prevented, thus restoring enzyme activity. Lysosomal storage disorders (LSDs) are suitable candidates for ASSC treatment, as the levels of enzyme activity needed to prevent substrate storage are relatively low. In addition, ASSCs are orally active small molecules and have potential to gain access to most cell types to treat neuronopathic LSDs. Competitive enzyme inhibitors are effective ASSCs when they are used at sub-inhibitory concentrations. This whole new paradigm provides excellent opportunity for identifying specific drugs to treat a broad range of inherited disorders. This review describes protein misfolding as a pathophysiological cause in LSDs and provides an overview of recent advances in the development of pharmacological chaperone therapy for the diseases. In addition, a generalized guidance for the design and screening of ASSCs is also presented.     

Photon enzyme activation

The electromagnetic molecular electronic resonance (EMER) frequencies of the molecular chains of α-chymotrypsin are calculated. The chain length relations and coupling positions suggest a possible energy transfer, at the EMER frequencies, from one chain to the other. Photon enzyme activation data indicate that the energy corresponding to the EMER frequencies of its molecular chains is used by α-chymotrypsin for its enzyme function.     

Regulation of enzyme activity by enzyme orientation: A hypothesis

In addition to substrate binding sites, many enzymes must possess supersubstrate binding sites that regulate attachment and orientation of the enzyme toward the matrix (micelle, membrane) in which the substrate molecules are embedded, the supersubstrate.     

Amperometric enzyme electrodes

Three different types of amperometric enzyme electrode are described. The first type uses a conducting organic-salt electrode to oxidize NADH. Results for sensors for ethanol and for bile acids are presented. In the second type of sensor, flavoenzymes are directly oxidized on the surface of the conducting organic-salt electrode. Results for five different enzymes are described. The mechanism of the enzyme oxidation is discussed and the reaction is shown to take place by heterogeneous redox catalysis and not by homogeneous mediation. The enzymes are strongly adsorbed on the electrode; microelectrodes for in vivo studies can be constructed without a membrane. Results for in vivo studies of changing glucose levels in the brain of a freely moving rat are presented. The third type of sensor is designed to measure low levels of toxic gases such as H2S and HCN. This is done by monitoring the inhibition by the toxic gas of the activity of the respiratory enzyme cytochrome oxidase.     

Industrial enzyme applications

The effective catalytic properties of enzymes have already promoted their introduction into several industrial products and processes. Recent developments in biotechnology, particularly in areas such as protein engineering and directed evolution, have provided important tools for the efficient development of new enzymes. This has resulted in the development of enzymes with improved properties for established technical applications and in the production of new enzymes tailor-made for entirely new areas of application where enzymes have not previously been used.