Substrate analog

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Substrate analogs, or transition-state analogs, are molecules that can be recognized and bound by an enzyme because they sufficiently resemble that enzyme's substrate. However, the enzyme is unable to affect the analog in the same manner it can affect its natural substrate. Thus substrate analogs can be efficient inhibitors of a given catalytic process and can provide unique opportunities for the study of biologically active molecules.

Applicable background

An enzyme is a molecule that binds a specific substrate and increases the rate at which the substrate is converted into a specific product. All substrates must pass though the transition state, a high energy conformation that normally prevents the spontaneous conversion of substrate to product. Most enzymes stabilize the transition state, thus lowering the free energy associated with it. This change in transition-state free energy increases the reaction rate according to the Arrhenius equation. Many enzyme inhibitors are substrate analogs.

Generalized mechanism

While an incredibly large number of non-substrate molecules may enter the active site of an enzyme, only a very small number of these, analogs, will be able to elicit affection from the enzyme. The specific mechanism varies from one analog to another, but the general ability for enzymatic analogs to do this stems from their structural similarity to the transition state of an enzyme's substrate. For example, the analog N-(Phosphonacetyl)-L-aspartate (PALA) differs from the natural transition state by only a few functional groups[1].

To mimic a substrate's transition state is no small task, however. It is not enough to simply possess a few functional groups in common with the substrate, as enzyme-substrate interaction often involves multiple points of contact.


Substrate analogs can have a variety of academic and medical applications. Analogs that act as a suicide inhibitor are useful in drug design, the most famous of which is penicillin (see Beta-lactam antibiotic for mechanism). Suicide inhibitor analogs have also made it possible to determine the structural features of various enzymes . When coupled with X-ray crystallography, bound analogs have provided crucial insight into the mechanisms of enzymes.

Substrate analogs have made it possible to visualize the short-lived conformational change in N-Acetyltransferase when it binds its substrate[2]. This highlights the academically compelling properties of substrate analogs; they resemble the natural substrate enough to bind and affect the enzyme, but not enough to be processed as the natural substrate would. This method of crystallography has become an indispensable resource in the study of changes in quaternary structure during enzymatic catalysis, provided the enzyme in question has a know substrate analog.

Analogs that have an effect on their target can be used to identify properties of the enzyme's primary structure. In the gut-bound protease chymotrypsin, studies with the radio-labeled substrate analog Tosyl phenylalanyl chloromethyl ketone (TPCK) has identified some of the catalytic amino acid residues. Acting as a suicide inhibitor, TPCK alkylates a critical residue and halts the function of chymotrypsin[3]. Additional investigation into the subject has suggested a mechanism and provided evidence for the function of additional residues[4][5]


  1. Collins KD, Stark GR (1971). "Aspartate Transcarbamylase: Interaction With The Transition State Analogue N-(Phosphonacetyl)-L-Aspartate". Journal of Biological Chemistry 246 (21): 6599-605
  2. Hickman AB, et al (1999). "The Structural Basis of Ordered Substrate Binding by Serotonin N-Acetyltransferase: Enzyme Complex at 1.8 A ̊ Resolution with a Bisubstrate Analog". Cell 97: 361-9
  3. Schoellmann G, Elliott S (1962). "Direct Evidence for the Presence of Histidine in the Active Center of Chymotrypsin". Biochemistry ? 252-?
  4. Prorok M, et al (1994). "Chloroketone Hydrolysis by Chymotrypsin and N-Methylhistidyl-57-chymotrypsin: Implications for the Mechanism of Chymotrypsin Inactivation by Chloroketones". Biochemistry 33: 9784-90
  5. Kreutter K, et al (1994). "Three-Dimensional Structure of Chymotrypsin Inactivated with (2S)-N-Acetyl-~-alanyl-~-phenylalanyl a-chloroethane: Implications for the Mechanism of Inactivation of Serine Proteases by Chloroketones". Biochemistry 33: 13792-800