Substrates、底物

Definition

A substrate is a molecule upon which an enzyme acts and gives rise to a product.

Discovery

Louis Pasteur Studied the fermentation of alcohol by yeast using sugar as substrate. In 1897, Eduard Buchner contributed the first demonstration of a complex biochemical process outside of a cell used yeast extracts to ferment sugar substrate, despite the absence of living yeast cells. In 1958, Daniel Koshland suggested that interaction of substrate to the active site of an enzyme creates an "induced fit" between enzyme and substrate. The amino acids side chains that make up the active site are molded into a precise shape, which enables the enzyme to perform its catalytic function. In some cases, the substrate molecule changes shape slightly as it enters the active site 1,2.  In 1913 Leonor Michaelis and Maud Menten explained the important relationship between rate of reaction, enzymes and substrate concentration 3.

Structural Characteristics

Emil Fischer in 1894 suggested that both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another.This is known as "the lock and key" model. This model explains enzyme specificity for the substrate 4. Daniel Koshland suggested induced fit model where substrate and enzymes are having rather flexible structures, the active site is continually reshaped by interactions with the substrate as the substrate interacts with the enzyme 5. They suggest that the substrate does not simply bind to a rigid active site but the amino acid side chains which make up the active site are induced into the precise position that helps the enzyme to perform its catalytic function 6. The active site continues to change until the substrate is completely fit, at which point the final shape and charge is determined . Several crystal structure of substrate-enzyme complex have been solved, X-ray crystal structures of methylmalonyl-CoA mutase in complexes with substrate methylmalonyl-CoA and inhibitors 2-carboxypropyl-CoA and 3-carboxypropyl-CoA (substrate and product analogues) show that the enzyme-substrate interactions change little during the course of the rearrangement reaction, in contrast to the large conformational change on substrate binding 7.

Mode of Action

Substrates are usually protein molecules that bind to enzymes. These substrate molecules bind to an enzyme's active site and are transformed into products through a series of steps known as the enzyme kinetics. Products of these reactions are released and the active enzyme reconstitutes. By increasing the substrate concentration, the rate of reaction will increase due to the likelihood that the number of enzyme-substrate complexes will increase; this occurs until the enzyme becomes the limiting factor. Most enzymatic transformations are reversible. These mechanisms can be divided into single-substrate and multiple-substrate mechanisms. In Single substrate reactions, the substrate binds with the enzyme active site leads to formation of enzyme-substrate complex. The substrate is transformed into one or more products, which are then released from the active site. The active site is now free to accept another substrate molecule. In enzymes with more than one substrate, substrates may bind in a particular order to the active site, before reacting together to produce products. Enzyme reactions involving two substrates and two products can proceed by at least three distinct mechanisms: random ordered, compulsory ordered, and double-displacement reactions. In many enzymatic reactions, and in particular biological reactions, a second substrate (i.e., species) must be introduced to activate the enzyme

Functions

Detectable signal, Enzyme substrates that produce a detectable signal upon transformation are used to measure enzymatic activity: detectable signals include changes in color, fluorescence or emission of light (luminescence). The corresponding substrates are called chromogenic, fluorogenic and luminogenic substrates.

Chemical industry, Substrate-Enzymes reactions are used in the chemical industry and other industrial applications although enzymes in general are limited in the number of reactions they have evolved to catalyze and also by their lack of stability in organic solvents and at high temperatures 8.

Industrial sanitation, Enzyme and enzyme substrates in combination can be used as reagents to assay co-factors in an enzymatic transformation such as ATP for the bioluminescence of luciferin eg, in the monitoring of industrial sanitation.

Clinical Diagnostics, Substrate-enzyme assays represent an important tool in clinical diagnostics. Immunoassays are used to detect a wide variety of metabolites associated with health condition. Immunoassays typically involve substrates for horseradish peroxidase (HRP) or alkaline phosphatase (AP), which are the standard markers of secondary antibodies in indirect ELISA assays

Molecular biology, Substrates and enzymes are an important tool used to facilitate cloning of DNA fragments. Substrates are also used to detect enzymatic activity in western, southern and northern blotting, as well as in reporter gene expression and in staining transgenic cells. Moreover, enzyme substrates can be utilized to assay co-factors of enzymatic reactions 9.

References

1.Briggs GE, Haldane JBS (1925). A note on the kinetics of enzyme action. Biochem. J., 19:339-339.

2.Cha Y, Murray CJ, Klinman JP (1989). Hydrogen tunneling in enzyme reactions. Science, 243:1325-1330.

3.Michaelis L, Menten M (1913). Die Kinetik der Invertinwirkung. Biochem. Z., 49:333-369.

4.Fischer E (1894). Einfluss der Configuration auf die Wirkung der Enzyme. Ber. Dt. Chem. Ges., 27:2985–2993.

5.Koshland DE (1958). Application of a Theory of Enzyme Specificity to Protein Synthesis. PNAS., 44(2):98–104.

6.Vasella A, Davies GJ, Böhm M (2002). Glycosidase mechanisms. Curr Opin Chem Biol., 6(5):619–629.

7.Mancia F, Smith GA, Evans PR (1999). Crystal Structure of Substrate Complexes of Methylmalonyl-CoA Mutase.  Biochemistry, 38(25):7999–8005.

8.Hult K, Berglund P (2003). Engineered enzymes for improved organic synthesis. Curr Opin Biotechnol., 14(4):395–400.

9.Saghatelian A, Trauger SA, Want EJ, Hawkins EG, Siuzdak G, and Cravatt BF (2004) Assignment of endogenous substrates to enzymes by global metabolite profiling. Biochemistry, 43(45):14332-14339.