Substrate analogues

Reversible inhibition is characterized by an equiUbrium between enzyme and inhibitor. Many reversible inhibitors are substrate analogues, and bear a close relationship to the normal substrate. When the inhibitor and the substrate compete for the same site on the enzyme, the inhibition is called competitive inhibition. In addition to the reaction described in equation 1, the competing reaction described in equation 3 proceeds when a competitive inhibitor I is added to the reaction solution. 

Polyalcohols, such as glycerol, sugar, sorbitol, and propylene glycol may prevent denaturation (28). Also substrates or substrate analogues often stabilize by conferring an increased rigidity to the enzyme stmcture. 

A recent example is the substrate analogue thymidine 5 -[a,P-iaiido]triphosphate [141171-20-2] (TMPNPP) (2) which competitively inhibits the human iaimunodeficiency vims-1 (HIV-1) reverse transcriptase (HIV-1 RT) with a iC value of 2.4 micromolar ]lM) (9). The substrate is thymidine 5 -triphosphate... 

Affinity Labels. Active site-directed, irreversible inhibitors or affinity labels are usually substrate analogues that contain a reactive electrophilic functional group. In the first step, they bind to the active site of the target enzyme in a reversible fashion. Subsequentiy, an active site nucleophile in close proximity reacts with the electrophilic group on the substrate to form a covalent bond between the enzyme and the inhibitor, typically via S 2 alkylation or acylation. Affinity labels do not require activation by the catalysis of the enzyme, as in the case of a mechanism-based inhibitor. 

Inhibition The decrease of the rate of an enzyme-catalyzed reaction by a chemical compound including substrate analogues. Such inhibition may be competitive with the substrate (binding at die active site of die enzyme) or non-competitive (binding at an allosteric site). 

Scheme 10.28 Reaction of HppE with substrate analogues.

Holzman, T. F., and Baldwin, T. O. (1982). Isolation of bacterial luciferases by affinity chromatography on 2,2-diphenylpropylamine-Sepharose phosphate-mediated binding to immobilized substrate analogue. Biochemistry 21 6194-6201. 

Matthews, J. C., Hori, K., and Cornier, M. J. (1977b). Substrate and substrate analogue binding properties of Renilla luciferase. Biochemistry 16 5217-5220. 

The first substrate analogue inhibitors of FAAH were reported in 1994. The anandamide analogues prepared represented three elasses of putative transition-state inhibitors a-trifluoromethyl ketones, a-ketoesters and a-ketoamides [62], In the initial sereening studies, it was found that the trifluoromethyl ketone eompounds tested were effeetive inhibitors of AEA hydrolysis. A selected set of a-keto esters also inhibited hydrolysis, while a-keto amides were ineffective. In particular, arachidonyl trifluoromethyl ketone (32), gave almost 100% inhibition of anandamide hydrolysis. A detailed investigation of the structural requirements for FAAH inhibition with a-trifluoromethyl ketones has been carried out by Roger and co-workers [63]. 

Finally, in the case of inhibitory substrate analogues such as allo-xanthine, strong evidence has recently been presented that these bind to molybdenum in reduced xanthine oxidase (33). If the enzyme is reduced with xanthine, then treated anaerobically with alloxanthine and finally exposed to air, catalytic activity is lost. Though flavin and iron in the final product are in the oxidized state, there are significant spectral differences between it and the native enzyme. These are believed (33) due to reduction of molybdenum from Mo(VI) to Mo(IV) and complexing of... 

Coan, C., Scales, D.J., and Murphy, AJ. 1996. Oligovanadate binding to sarcoplasmatic reticulum ATPase evidence for substrate analogue behaviour. Journal of Biological Chemistry 261 10394—10403. 

Adams, P.R., Harrison, R., and Inch, T.D., Dehydrogenation of a phosphonate substrate analogue by glycerol 3-phosphate dehydrogenase, Biochem.., 141, 729, 1974. 

Fig. 5. View of PLCBc bound to a phosphonate substrate analogue showing protein backbone (cyan), zinc ions (magenta), and inhibitor with carbons (white), oxygens (red), phosphorus (orange), and nitrogen (blue) [45]... 

Fig. 8a, b. Proposed modes of substrate binding to a PLCBc [45] b PI nuclease [70], based on X-ray structures with non-hydrolyzable substrate analogues... 

Fig. 9. Stabilizing interactions of trimethylammonium ion of a substrate analogue with Glu4, Tyr56, and Phe66 of PLCa. [45]...

Fig. 12. Distances (A) of PLCBc active site waters from selected amino acid side chains and a phosphonate substrate analogue [45]...

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