Studies Of Enzyme Catalysis Research Articles

Chemical Synthesis of an Enzyme Containing an Artificial Catalytic Apparatus

With the goal of investigating electronic aspects of the catalysis of peptide bond hydrolysis, an analogue of HIV-1 protease was designed in which a non-peptide hydroxy-isoquinolinone artificial catalytic apparatus replaced the conserved Asp25–Thr26–Gly27 sequence in each 99-residue polypeptide chain of the homodimeric enzyme molecule. The enzyme analogue was prepared by total chemical synthesis and had detectable catalytic activity on known HIV-1 protease peptide substrates. Compared with uncatalyzed hydrolysis, the analogue enzyme increased the rate of peptide bond hydrolysis by ∼108-fold. Extensions of this unique approach to the study of enzyme catalysis in HIV-1 protease are discussed.

Single‐Molecule Studies of HIV‐1 Protease Catalysis Enabled by Chemical Protein Synthesis

AbstractSingle‐molecule fluorescence studies of the proteolytic activity of the enzyme HIV‐1 protease were performed using FRET‐pair dye labeled peptide substrates and substrate‐derived inhibitors prepared by solid phase peptide synthesis. Chemical protein synthesis was used to prepare homodimeric HIV‐1 protease in soluble form and to prepare a covalent dimer 203 amino acid residue HIV‐1 protease containing a biotin tether at the mid‐point of the synthetic protein molecule. The biotin‐tagged HIV‐1 protease was immobilized on a neutravidin‐coated glass slide. Total internal reflection excitation multiwavelength fluorescence spectroscopy was used to monitor substrate binding and cleavage by the synthetic enzyme molecules. Single‐molecule traces for the dye‐labeled peptide substrate showed distinct binding and cleavage events; the corresponding dye‐labeled peptide inhibitor showed only the binding event. These results constitute strong proof‐of‐principle for the utility of chemical peptide and protein synthesis for single‐molecule studies of enzyme catalysis.

An experiment on the chemical modification of essential tryptophan residues of barley β-amylase

Introduction Enzymes bind their substrates through a specific limited area on the enzyme surface called the active site at which catalysis occurs. Apart from X-ray diffraction, the most successful approach to the study of the components and the threedimensional conformation of the active site is of modification of putative essential residues by group-specific chemical reagents, affinity labels etc. Small alterations in the structure may be detected from the reactivity of amino acid residues with specific reagents. Modification studies can give a better understanding of the catalytic mechanism or detect which amino acid residues are directly involved in the catalytic process. Kinetic studies together with chemical modification can give a more complete picture of the active site amino acids. As a complement to the study of enzyme catalysis and kinetics, UV-difference spectra can highlight the implication of certain essential residues in the active site. We have chosen barley 13-amylase (BA) whose activity can be measured very easily. I BA contains essential tryptophan residues which may be chemically modified by treatment with Nbromosuccinimide (NBS). These experiments described are used in a biochemistry practical session at the Department of Biochemistry, Gulbarga University at first-year MSc (Pre) level (intake 15 students a year). Another consideration is that the student should be able to complete the practical within 3-4 hours. The practical described here is a simple and straightforward demonstration of the various aspects of enzymes.

Self-directed study of enzyme catalysis using computer and print resources

Self-directed study of enzyme catalysis using computer and print resources

Equilibrium Kinetic Studies of Enzyme Mechanism and Control

This chapter discusses equilibrium kinetic studies of enzyme mechanism and control. It focuses on some aspects of the isotopic exchange kinetics at equilibrium and its evolution from the study of enzymic catalysis by the manipulation of reactants to the study of the control of catalysis by ligands that are not, themselves, catalytically transformed, inhibitors and activators, particularly of allosteric enzymes. The chapter presents particular areas of usefulness of these measurements with some examples. An important theroretical use of isotopic exchange at equilibrium was suggested to be the elucidation of compulsory pathways or binding orders. The usefulness of isotopic exchange kinetics for elucidation of compulsory binding orders was first established for the bovine heart and rabbit muscle lactate dehydrogenases. These enzymes were believed to have a compulsory pathway on the basis of binding and kinetic data. Subsequently, a compulsory pathway was demonstrated for porcine and bovine heart malate dehydrogenases and bovine liver glutamate dehydrogenase at pH 8.8. The study of enzyme catalysis at equilibrium by isotopic exchange of reactants is useful for elucidation of aspects of enzyme mechanism which may not be readily obtainable by other means. These include elucidation of a compulsory binding order, indications of fast and slow dissociation steps and whether chemical transformation is rate limiting; determination of the possibility of abortive complex formation, and, importantly, the observation of non-rate-limiting steps in catalysis. Another important aspect of equilibrium kinetics is its extension to the study of the mechanism of modifiers of enzymic catalysis, particularly with respect to effectors of allosteric enzymes.

Metalloenzymer and model systems. Carbonic Anhydrase: Solvent and Buffer Participation, isotope effects and activation parameters

It is a general dilemma in biophysical chemistry that although small perturbations are theoretically most desirable, they tend to produce small effects which in turn are difficult to interpret, especially when systems are as large and complex as those encountered in the study of enzyme catalysis. In this regard the replacement of H 2O is slowly becoming a powerful tool, especially in hydrationdehydration studies, because it gives measurable results from a modest perturbation of those molecular properties which are sensitive ot mass. The ubiquity of CO 2 has prompted us to investigate one of life's most fundamental reactions, the reversible hydration of CO 2. We find that many general bases, and especially meta ion-hydroxo complexes catalyze this reaction, but the most potent of these catalysts is the zinc metalloenzyme carbonic anhydrase (carbonate hydro-lyase EC 4.2.1.1) from red blood cells. The mammalian enzyme is especially versatile catalyzing not only the reversible hydration of CO 2 but also that of aliphatic aldehydes, pyridine carboxaldehydes, pyruvic and alkyl pyruvate esters. The enzyme is also an efficient esterase, catalyzing the hydrolysis of certain carboxylate, sulfonate, phosphate and imidate esters. Substitution of zinc(II) by cobalt(II) gives an active enzyme which is being studied by observing its response to perturbations induced by substrates, buffer and inhibitors. We have recently studied the effects of pH, buffer concentration, steric hindrance, solvent composition (H 2O vs. D 2O) and inhibitor binding on CO 2 hydration, HCO − 3 dehydration and 18O exchange in the CO 2HCO − 3 system at four temperature ranging from 7° to 35 °C. Our findings allow us to eliminate a number of anomalous mechanisms pertaining to isotopic exchange and provide us with a suitable framework for transition state characterization. Furthermore, our work shows that the Haldane relation is obeyed at every pH(pd), buffer and inhibitor concentration studied. Compliance with this relation shows that the system meets all the criteria required for the attainment of a chemical equilibrium.

13] Cryoenzymology: The study of enzyme catalysis at subzero temperatures

13] Cryoenzymology: The study of enzyme catalysis at subzero temperatures