Oxyanion complexes, stability

X-ray crystallographic studies of serine protease complexes with transition-state analogs have shown how chymotrypsin stabilizes the tetrahedral oxyanion transition states (structures (c) and (g) in Figure 16.24) of the protease reaction. The amide nitrogens of Ser and Gly form an oxyanion hole in which the substrate carbonyl oxygen is hydrogen-bonded to the amide N-H groups. 

For many serine and cysteine peptidases catalysis first involves formation of a complex known as an acyl intermediate. An essential residue is required to stabilize this intermediate by helping to form the oxyanion hole. In cathepsin B a glutamine performs this role and sometimes a catalytic tetrad (Gin, Cys, His, Asn) is referred too. In chymotrypsin, a glycine is essential for stabilizing the oxyanion hole. 

Kim, Chin, and co-workers have described a highly interesting oxyanion hole mimic that transforms L-amino acids to D-amino acids [97]. The mechanism involves stabilization of the enolate intermediate by an internal hydrogen bond array generated by urea group (Scheme 4.14). In the presence of an external base, such as triethylamine, the receptors readily promote the epimerization of a-amino acids, favoring the D-amino acids due to unfavorable steric interactions in the receptor-L-amino acid complex. These receptors can also be viewed as chiral mimics of pyridoxal phosphate [98]. 

The (5)-tryptophan-derived oxazaborolidenes utilized in this aldol study have been previously examined by Corey as effective catalysts for enantioselective Diels-Alder cycloaddition reactions [6]. Corey has documented unique physical properties of the complex and has proposed that the electron-rich indole participates in stabilizing a donor-acceptor interaction with the metal-bound polarized aldehyde. More recently, Corey has formulated a model exemplified by 7 in which binding by the aldehyde to the metal is rigidified through the formation of a hydrogen-bond between the polarized formyl C-H and an oxyanionic ligand [7], The model illustrates the sophisticated design elements that can be incorporated into the preparation of transition-metal complexes that lead to exquisite control in aldehyde enantiofacial differentiation. 

Differences in the speciation of other ions at the surface can be noted. Using an analysis similar to that above for metal ions, one finds that adsorbed anions are less acidic than in bulk solution. For example, it was shown in Figure 6 that protolysis of adsorbed sulfate ions becomes significant in the pH range 4-5, whereas in solution bisulfate is formed at much more acidic conditions (pH 2). Complexes formed by supporting electrolyte, e.g. Na" ,. with oxide surface sites have greater stability constants (logK 0.5-1.7) than observed for complex formation with oxyanions in solution (log K 0.0) (J.). ... 

Antibody esterase 48G7 was elicited against hapten 1 and effectively catalyzed the hydrolysis of the corresponding activated ester 2 (27). The X-ray crystal structure of this catalytic antibody Fab complexed with 1 revealed the corresponding stabilization of the oxyanion by a nearby cationic Arg residue (27, 38). Hydrogen bonds from the side chains of the adjacent amino acids His and Tyr serve to stabilize the polarized phosphoryl bonds of hapten 1 that would assist in forming the transition state of ester 2. Main-chain amide bonds from Tyr and Tyr also provide additional hydrogen-bond stabilization forces. 

Circiunstantial support for this mechanism was supplied by the fact that A-tosyl-Phe-CMK, a specific inhibitor of chymotrypsin, did not react with anhydrochymotrypsin [104]. Although both X-ray crystallographic and NMR studies supported the alkylated hemiketal as the structure of the inhibited enzyme, those studies did not prove whether alkylation or hemiketal formation oecurred first [105, 98]. Carbon-13 NMR studies were also used to determine the pKa (7.88-8.1) of the hemiketal hydroxyl and this finding provided the first evidence that serine proteinases could stabilize the ionized form of the alkylated hemiketal, via hydrogen bonds in the oxyanion hole [106,107]. A series of more recent papers has confirmed that hemiketal formation precedes the alkylation step and has shown that the initial, reversible part of the interaction is made up of two discrete stages (a) formation of a Michaelis complex, followed by (b) hemiketal formation [102, 108]. The requirement of an intermediate hemiketal may mean that chloromethyl ketone (CMK) inhibitors should be considered as transition-state [109] analogue inhibitors (see diseussion in seetion on Aldehydes). 

X-ray structures have been worked out for the benzeneboronic and 2-phenyl-ethaneboronic acid (PEBA) complexes of subtilisin [9] and for the PEBA complex of a-chymotrypsin (a-CHT) dimer [10]. Further stabilization of the hydroxyls on boron is gained by hydrogen bonding to other amido groups lining the oxyanion hole. 

---