Studies of Enzyme Mechanism Active Site

1 Chemical modifications. Despite many obvions limitations, chenucal modification had been the easiest approach to explore essential groups at the active site of an enzyme. However, chenucal modification has been largely replaced by the 

Amino acid residues, except hydrocarbon chains, may provide nucleophilic sites (electron-rich centers) or electrophilic sites (electron-deficient centers) for chemical modifications. Electron-rich centers include sulfur nucleophiles (thiol of Cys and thioether of Thr), nitrogen nucleophiles (e-amino of Lys, imidazole of His and Guanidyl of Arg), oxygen nucleophiles (phenolic of Tyr, carboxyl of Asp and Glu and hydroxyl of Ser and Thr), and carbon nucleophile (a-position of indole ring of Trp), with an increasing nucleophilicity in that order. They provide nucleophilic sites for alkylation (nncleophilic substitution), acylation, addition and oxidation at pH near or above their pK values. Electron-deficient centers include ammonium cation of Lys, guanidiiun cation of Arg and imidazolium cation of His. They provide electrophilic sites for metalation and reduction at pH near or below their pK values. 

The chemical properties of a protein fimctional group are strongly influenced by its local environment, such as polarity of the microenvironment, hydrogen bonding effect, field/electrostatic effect, as well as steric and matrix effects. These factors contribute to the selectivity and specificity of chemical modifications. Several strategies can be adapted to enhance the selectivity and specificity  

the exploitation of enhanced or differential reactivity of amino add residues  

the exploitation of protein specificity such as affinity labeling (Wold, 1977) and 

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