Enzymes active site probing

Cr(OH2)4(thionucleotide)]"" Chiral, exchange-inert, enzyme active site probes 154... 

L. J. Mueller and M. F. Dunn, NMR Crystallography of Enzyme Active Sites Probing Chemically Detailed, Three-Dimensional Structure in Tryptophan S mthase, Acc. Chem. Res., 2013, 46, 2008. 

Substrate analogs which promise to be particularly good active-site probes are those which are conformationally restricted. One key feature of enzymatic processes is that when a substrate is bound to an enzyme, probably only one of the many possible conformations of the substrate molecule is assumed. Consequently, before a detailed mechanism for an enzymatic process can be formulated, the preferred conformations of each of the enzyme-bound substrates must be known. ... 

Various strains of oral streptococci produce D-glucosyltransferases which utilize sucrose as a o-glucosyl donor in the production of soluble and insoluble D-glucans. Consequently, it may be expected that some deoxyfluoro derivatives of sucrose function as competitive inhibitors for the dextransu-crases of tooth bacteria, thus preventing decay, or at least may be used as active-site probes for the enzymes. Another aim of these researches is to find non-metabolizable sweeteners. 

The final method of coupling enzyme reactions to electrochemistry is to immobilize an enzyme directly at the electrode surface. Enzyme electrodes provide the advantages already discussed for immobilization of enzymes. In addition, the transport of enzyme product from the enzyme active site to the electrode surface is greatly enhanced when the enzyme is very near to the electrode. The concept of combining an enzyme reaction with an amperometric probe should offer all of the advantages discussed earlier for ion-selective (potentiometric) electrodes with a much higher sensitivity. In addition, since the response of amperometric electrodes is linear, background can be selected. 

Activity-based protein profiling (ABPP) is a chemical proteomic strategy in which active-site-directed covalent probes are used to profile the functional states of enzymes in complex proteomes. Activity-based probes (ABPs) can distinguish active enzymes from their inactive zymogens or inhibitor-bound forms. They contain a reactive group intended to modify enzyme active sites covalently and a reporter group (typically rhodamine or biotin) that assists in detection and identification of protein targets. 

A cyclohexadienyl Lewis adduct or salt formed by the reaction of a Lewis base with an aromatic compound. Such an adduct is apparently formed from the reaction of OH with 4-(A/-2-aminoethyl-2 -pyridyl disulfide)- -nitrobenzo-2-oxa-l,3-diazole (2PROD). 2PROD is a two-protonic-state electrophile used as a probe for enzyme active site nucleophiles and as a fluorescent re-... 

Webb, M. E., Stephens, E., Smith, A. G and Abell, C. (2003). Rapid screening by MALDI-TOF mass spectrometry to probe binding specificity at enzyme active sites. Chem. Commun., 2416-2417. 

The principle advantage of the physical labeling method is the possibility of receiving direct information about the structure, mobility and local micropolarity of certain parts of a molecular object of any molecular mass. Developments in synthetic chemistry, biochemistry and site-directed mutagenesis have provided researchers with a wide assortment of labels and probes, and have paved the way for the specific modification of protein function groups, including enzyme active sites. 

Figure 10. Structures of electron-transfer probes used to examine the chemistry of enzyme active sites which are deeply buried within the protein, in particular the cytochrome P450 [354].

Phosphonamidothioates show strong promise as potent tetrahedral-inter-mediate analogue inhibitors of metallopeptidases, with the unique value of probing enzyme active site architecture with complementary chiral phosphorus... 

Carba-sugars are usually recognized by enzymes involving their parent sugars, but do not have sufficient affinity to act as efficient inhibitors. For this purpose, additional features such as amino groups or unsaturation are needed for the molecule to mimic a transition state, rather than a substrate. However, carba-sugars have been successfully used as probes to elucidate biosynthetic pathways and the topology of enzyme active sites. 

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