Acyl Transpeptidation and Hydrolytic Reactions Catalyzed by Endothia Parasitica Protease With Small Peptide Substrates

Abstract

Endothia parasitica protease shows hysteretic behavior (a lag phase) when acting upon L-leucyl-L-leucine amide at pH 4.5. Initially, the products are L-leucyl-L-leucine, L-leucyl-L-leucyl-L-leucine and L-leucine amide with little formation of L-leucine and no ammonia. The relative concentration ofL-leucyl-L-leucine is about one-fourth that ofL-leucine amide. As the reaction continues the relative concentration of L-leucine increases slowly until it is equal to the concentration of L-leucyl-L-leucine near the end of the reaction with each being one-fourth that of L-leucine amide. Formation of these products can only be explained by a combination of transpeptidation and hydrolytic reactions. By itself, L-leucyl-L-leucine is cleaved very slowly but when mixed with L-leucyl-L-leucine amide the rate is several fold greater than the combined rates of the two alone. L-Leucyl-L-leucyl-L-leucine and L-leucyl-L-leucyl-L-leucine amide as substrates of E. parasitica protease give normal kinetic behavior with no evidence of formation of higher molecular weight compounds. Addition of as little as 1 pM L-leucyl-L-leucyl-L-leucine (Km = 0.348 mM) to a reaction containing 1 mM L-leucyl-L-leucine amide increases the rate of reaction with a shortening of the lag phase. Addition of 100 pM L-leucyl-L-leucyl-L-leucine to such a reaction eliminates the hysteretic behavior. It is proposed that E. parasitica protease is initially in an inactive form which can be activated by substrates as small as L-leucyl-L-leucyl-L-leucine but not by L-leucyl-Lleucine amide. During the lag phase observed with substrates such as L-leucyl-L-leucine amide, the enzyme produces, via my1 transpptidation, larger compounds which can rapidly activate the enzyme. While in the activated form, E. parasitica protease cleaves L-leucyl-L-leucine amide via normal kinetic behavior; however, both acyl transpptidution and hy-53 54 SHIH-LO LEE AND JOHN R. WHITAKER drolysis still occur as shown by the relative concentrations of L-leucine, L-leucyl-L-leucine and L-leucine amide of 1:1:4 near end of the reaction. E. parasitica protease cannot cleave carbobenzoxy-L-leucyl-L-leucine indicating that, at least with this substrate, the enzyme cannot perform amino transpeptidation reactions.CD spectra were measured on a Cary 60 spectrometer using a cuvette with light path of 1 cm and a full range of ellipticity of 0.02 degree. The signal output was fed to a PDPSE computer which averaged the signal and printed out the readings at intervals of one nm (with 10% error). The CD spectrum of the buffer (0.1 M sodium acetate buffer, pH 4.51, taken first, was subtracted from the spectrum of the enzyme in solution automatically by the computer. The spectrum of E. parasitica protease was taken at a final concentration of 20 pM in a volume of 3 ml in a cylindrical cuvette in the presence and absence of Leu-Leu amide.