Enzyme unit

Abstract: The three-dimensional structure of the enzyme dihydrodipicolinate synthase (KEGG entry Rv2753c, EC 4.2.1.52) from Mycobacterium tuberculosis (Mtb-DHDPS) was determined and refined at 2.28 A (1 A=0.1 nm) resolution. The asymmetric unit of the crystal contains two tetramers, each of which we propose to be the functional enzyme unit. This is supported by analytical ultracentrifugation studies, which show the enzyme to be tetrameric in solution. The structure of each subunit consists of an N-terminal (beta/alpha)(8)-barrel followed by a C-terminal alpha-helical domain. The active site comprises residues from two adjacent subunits, across an interface, and is located at the C-terminal side of the (beta/alpha)(8)-barrel domain. A comparison with the other known DHDPS structures shows that the overall architecture of the active site is largely conserved, albeit the proton relay motif comprising Tyr(143), Thr(54) and Tyr(117) appears to be disrupted. The kinetic parameters of the enzyme are reported: K(M)(ASA)=0.43+/-0.02 mM, K(M)(pyruvate)=0.17+/-0.01 mM and V(max)=4.42+/-0.08 micromol x s(-1) x mg(-1). Interestingly, the V(max) of Mtb-DHDPS is 6-fold higher than the corresponding value for Escherichia coli DHDPS, and the enzyme is insensitive to feedback inhibition by (S)-lysine. This can be explained by the three-dimensional structure, which shows that the (S)-lysine-binding site is not conserved in Mtb-DHDPS, when compared with DHDPS enzymes that are known to be inhibited by (S)-lysine. A selection of metabolites from the aspartate family of amino acids do not inhibit this enzyme. A comprehensive understanding of the structure and function of this important enzyme from the (S)-lysine biosynthesis pathway may provide the key for the design of new antibiotics to combat tuberculosis.

Abstract: Publisher Summary This chapter presents a procedure for purification and assay of mast cell proteases. For Purification procedure mast cells are collected from Sprague-Dawley rats by washing the peritoneal cavities with 10 ml of ice-cold phosphate-buffered saline solution at pH 7.2. All subsequent steps are carried out at 4°C, including affinity-adsorption chromatography and adsorption to barium sulfate. During the purification of the mast cell protease, it is important to maintain the solutions at relatively high ionic strength to prevent the enzyme from adsorbing to surfaces. The assay measures chymotrypsin-like esterase activity. The procedure involves mixing of substrate and diluted enzyme appended with the buffer. The absorption change at 256 nm is recorded for about 5 rain. An enzyme unit is defined as an amount of enzyme activity that results in the hydrolysis of 1 μmol of substrate per minute at pH 7.8, 25°C.

Abstract: Publisher Summary Microsomal epoxide hydrolase catalyzes the conversionof epoxides to glycols. The microsomal enzyme should not be confused with another epoxide hydrolase activity, found primarily in the cytosolic fraction, which differs greatly from membrane-bound enzyme in substrate specificity and immunological properties. As with other membrane-bound enzymes, it is highly lipophilic and easily forms aggregates in solution. Its physical properties, therefore, present special problems for purification. The general scheme of purification presented in this chapter consists of solubilization from microsomes by a nonionic detergent; chromatography with siethylaminoethyl-cellulose and phosphocellulose, which effect purification on the basis of charge; chromatography with butylsepharose, which effects purification on the basis of hydrophobicity; and the removal of detergent by a second phosphocellulose step. Enzyme activity can be assayed by the conversion of radiolabeled epoxide substrates to dihydrodiols and the subsequent separation of products from substrate by simple solvent extraction. Tritiated benzo[a]pyrene 4,5-oxide and styrene 7,8- oxide can be used to monitor enzyme activity during purification. One enzyme unit is defined as the amount catalyzing the hydration of 1 pmol of styrene oxide in 1 min under specific conditions.