Chymotrypsin

While catalysis by aspartic proteases involves the direct hydro lytic attack of water on a peptide bond, catalysis by the serine protease chymotrypsin involves formation of a covalent acyl-enzyme intermediate. A conserved seryl residue, serine 195 in the bovine form, is activated via interactions with histidine 57 and aspartate 102. While these three residues are far apart in primary structure, in the active site of the mature, folded protein they reside within bond-forming distance of one another. Aligned in the order Asp 102-His 57-Ser 195, this trio forms a linked charge-relay network that acts as a “proton shuttle.”

Binding of substrate initiates proton shifts that in effect transfer the hydroxyl proton of Ser 195 to Asp 102 (Figure 1). The enhanced nucleophilicity of the seryl oxy gen facilitates its attack on the carbonyl carbon of the peptide bond of the substrate, forming a covalent acyl-enzyme inter mediate. The proton on Asp 102 then shuttles via His 57 to the amino group liberated when the peptide bond is cleaved. The portion of the original peptide with a free amino group then leaves the active site and is replaced by a water molecule. The charge-relay network now activates the water molecule by withdrawing a proton through His 57 to Asp 102. The resulting hydroxide ion attacks the acyl-enzyme intermediate, and a reverse proton shuttle returns a proton to Ser 195, restoring its original state. While modified during the process of catalysis, chymotrypsin emerges unchanged on completion of the reaction. The proteases trypsin and elastase employ a similar catalytic mechanism, but the numbering of the residues in their Ser-His-Asp proton shuttles differ.

Fig1. Catalysis by chymotrypsin. ➀ The charge-relay system removes a proton from Ser 195, making it a stronger nucleophile. ➁ Activated Ser 195 attacks the peptide bond, forming a transient tetrahedral intermediate. ➂ Release of the amino terminal peptide is facilitated by donation of a proton to the newly formed amino group by His 57 of the charge-relay system, yielding an acyl Ser 195 intermediate. ➃ His 57 and Asp 102 collaborate to activate a water molecule, which attacks the acyl-Ser 195, forming a second tetrahedral intermediate. ➄The charge-relay system donates a proton to Ser 195, facilitating breakdown of the tetrahedral intermediate to release the carboxyl terminal peptide ➅.

Fructose-2,6-Bisphosphatase

 Fructose-2,6-bisphosphatase, a regulatory enzyme of gluconeogenesis, catalyzes the hydrolytic release of the phosphate on carbon 2 of fructose-2,6-bisphosphate. Figure 2 illustrates the roles of seven active site residues. Catalysis involves a “catalytic triad” consisting of one Glu and two His residues, of which one His forms a covalent phosphohistidyl intermediate.

Fig1. Catalysis by fructose-2,6-bisphosphatase. (1) Lys 356 and Arg 257, 307, and 352 stabilize the quadruple negative charge of the substrate by charge–charge interactions. Glu 327 stabilizes the positive charge on His 392. (2) The nucleophile His 392 attacks the C-2 phosphoryl group and transfers it to His 258, forming a phosphoryl-enzyme intermediate. Fructose-6-phosphate now leaves the enzyme. (3) Nucleophilic attack by a water molecule, possibly assisted by Glu 327 acting as a base, forms inorganic phosphate. (4) Inorganic orthophosphate is released from Arg 257 and Arg 307. (Reproduced with permission from Pilkis SJ, Claus TH, Kurland IJ, et al: 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase: a metabolic signaling enzyme, Annu Rev Biochem. 1995;64:799-835.)

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