A Comparative Study of Spin-labeled Serine Enzymes: Acetylcholinesterase, Trypsin, α-Chymotrypsin, Elastase, and Subtilisin

Acetylcholinesterase, trypsin, α-chymotrypsin, elastase, and subtilisin have been spin-labeled, each at its active site serine residue, by 1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl-methylphosphonofluoridate. The mobility of the label when attached to these enzymes falls into three categories: high mobility, acetylcholinesterase; moderate mobility, α-chymotrypsin; and low mobility, elastase and subtilisin. The mobility of the label attached to trypsin depends upon the pH at which the reaction is carried out; it is mobile when labeled at pH 5.5 and immobile when labeled at pH 7.7 Spin-labeled subtilisin undergoes rapid hydrolytic release of the phosphonyl nitroxide above pH 4.3, and experiences unfolding of the active site region below pH 3.5. These results are discussed in terms of the crystallographic data presently available for these enzymes.