Cholesterol Esterase

Cholesterol esterase (CHE, EC 3.1.1.13) is an important steroidal enzyme that catalyzes the hydrolysis of cholesterol esters into cholesterol and fatty acids. It is one of the important enzymes for the clinical detection of total serum cholesterol.

Sources

Cholesterol esterase is found in many biological tissues, including liver, pancreas, adrenal gland, gonad, breast, brain and corpus luteum. Cholesterol esterase products mainly came from the animal organs until the 1960s, while in recent years, many researchers pay attention to the production of cholesterol esterase by microorganisms. These microorganisms include pseudomonas fluorescent, pseudomonas mendocina, Alcaligenes sp., strptomyces lavendulac, fusarium oxysporum, sstrptomyces sp., rhodococcus.sp. et al. In 1975, Uwajima et al. firstly purified cholesterol esterase from fluorescent pseudomonas cultures. It has been known that different sources of cholesterol esterase may have different functions, and now cholesterol esterase in pancreatic, liver and light base were widely studied and used.

Characteristics

The characteristics of cholesterol esterase from different sources are different in molecular size, structure, substrate specificity and reaction kinetics. Generally, the relative molecular mass of CHE is in the range of 12kDa to 60kDa. However, the microbe-derived mammalian CHE has relatively large molecular weight (>60kDa), which composed of more than 500 amino acid residues. The molecular weight through gel filtration detection may be a little higher, which may be because of the formation of cholesterol acetase polymerase. According to the structural characteristics and the amino acid sequence of the enzyme, CHE can be divided into two group. CHE from higher eukaryotes, yeast, gram-negative bacteria sources constitutes a family, which have the same enzyme catalytic center contains a GXSXG sequence and a catalytic triplet, namely serine/histidine/aspartic and glutamic acid. The catalytic mechanism of CHE hydrolysis is familiar as the chymotrypsin serine protease. The cholesterol esterase from the actinomycetes constitutes another family, this family does not have the typical GXSXG sequence of the alcohol esterase, but has three conserved amino acid residues: two serine and one aspartic acid residue.

Structure

The crystal structure of cholesterol esterase derived from preudomonas sp. can be seen in Figure 1.The enzyme composed of two identical subunits, with a molecular weight of 63kDa, 534 amino acid residues. The characteristic of the CHE is that it contains 18 α-spirals,19 β-folds, and 33 lysine.

Figure 1. Cholesterol Esterase Crystal Structure at 1.4Å resolution (Pletnev, V. et al. 2002)

The cholesterol esterase structure is a dimer with four spatially separated interfacial contact areas and two symmetry-related pairs of openings to an internal intradimer cavity. Hydrophobic active-site gorges in each subunit face each other across a central interfacial cavity. The CHE subunits have carbohydrate chains attached to their Asn314 and Asn351 residues, with two ordered N-acetyl-D-glucosoamine moieties visible at each site.

Activity Assay

In recent years, researchers studied the substrate specificity and reaction kinetics of cholesterol esterase from microbial sources. There are some differences among the different resources of CHE, including pH, temperature, stability, substrate specificity and activity assay. The enzyme activity of CHE is determined based on the absorbance value measured at 500 nm. The determination of CHE activity can be concluded below: 1mL of liquid A and 500mL of liquid B, then adding 50μL of diluted properly deal with enzyme liquid, and react at 40°C accurately for 15 min. After that, add 1.4 mL 0. 1mol/L HCl to terminate the enzyme reaction. The enzyme activity was defined as the required enzyme amount for catalytic hydrolyzing 1μM substrate per minute.

CHE and Human Metabolism

Many researches have been built to understand the relationship between cholesterol esterase and human metabolism. It is known that pancreatic cholesterol esterase has substrate specificity for tril-, di-, and single phthalein and phospholipids. In the liver, it maintains the dynamic balance of cholesterol. Cholesterol has two forms in the human body, free and esterify. It is reported that CHE exist in liver lysosomes, cellular solutes and microsomes. The lysosomal acidic lipase, involving in the hydrolysis of lipoprotein cells during the transfer of cholesterol esters and triphthalein glycerol to hepatocellular receptors. The modification of cholesterol esterase is believed to play an important role in the pathogenesis of atherosclerosis. However, details of the activation and inhibition of cholesterol esterase still unknown.

Reference

1. Pletnev, V.; et al. Three-dimensional structure of homodimeric cholesterol esterase-ligand complex at 1.4 Å resolution. Acta Crystallogr. D. 2010. 59(1).