Chemical modification of carboxyl groups in porcine pepsin

Abstract

Carboxyl groups in porcine pepsin were chemically modified "by the carbodiimide reaction using waterrsoluble l-ethyl-3-(3-dimethylaminopropyl) carbodiimide and amino acid esters as nucleophiles. The modification resulted in profound changes in the activities, specificity and.some physicochemical properties of the enzyme. These include* (1) significant decrease in milk clotting activity without changes in proteolytic activity against hemoglobin; (2) decrease in peptidase activity against N-acetyl-L-phenylalanyl-diiodo-L-tyrosine; (3) increase in clotting activity against Xcasein but decrease in clotting activity against Kcasein mixture; (k) shift-in proteolytic pH profile with pH optimum increased from 2.0 to about 3*5; (5.) decrease in relative electrophoretic mobility and a slight decrease in isoelectric point; (6) increase in K m without much change in k cai; ; and (7) increase in stability at pH above 6.0. Results suggest that the drop in milk clotting activity was due to a change in the charge distribution on the enzyme affecting enzyme-micelle interaction. The presence of dipeptide substrates interfered with the carboxyl modification suggestive of the proximity of the modified groups to the enzyme active site. iii The modified enzyme remained reactive to site-specific inactivators but at rates slower than the native enzyme. The modification was not specific, causing similar changes in pepsinogen and chymosin. The modified and native pepsins had similar caseinolytic properties and produced comparable rates of syneresis and curd tension development on curdled milk. The increase in pH stability suggested that the modified enzyme may be a better calf rennet substitute than native pepsin for cheesemaking. of Pepsin .<>»<.

Reduction of negative charge in the aspartyl proteinase from the fungus Mucor miehei by chemical modification of carboxyl groups: effect on structure-function

“…Coomassie Brilliant Blue G-250 stained gels were scanned with a Zenith Soft-Laser densitometer (Model SLR-504-XL, Biomed Instruments, Fullerton, CA) to determine relative electrophoretic mobility of the bands; native MMP was assigned a relative value of 1.00. Agarose gel electrophoresis was performed since the use of low gel concentration (e.g., 1%) and resultant large pore size minimized molecular seiving and allowed separation of proteins based primarily on charge (Shaw, 1969;Ma and Nakai, 1980). Differential scanning calorimetry (DSC) measurements were made on a Du Pont differential scanning calorimeter Model 2910 and Thermal Analyst 2000 System (E. I. du Pont de Nemours and Co., Wilmington, DE) at a heating rate of 2 "C/min from 25 to 100 "C. The instrument was calibrated with indium at the specified heating rate.…”

“…Modification of Carboxyl Groups. Carboxyl groups in purified MMP were modified according to the carbodiimidemediated amide formation procedure of Hoare and Koshland (1967), as modified by Ma and Nakai (1980). The procedure involved activation of protein carboxyl groups, i.e., carboxyl side chains of aspartic and glutamic acids, by water-soluble EDC subsequent to nucleophilic attack by an amino acid methyl ester.…”

“…Ma and Nakai (1980) reported that chemical modification of carboxyl groups in porcine pepsin improved the stability of the enzyme near neutral PH. Yada and Nakai (1986a) proposed that the high MC/ PA of chymosin may be related to its high hydrophobicity and low charge.…”

“…More directly, nonselective chemical modification, i.e., modification of a specific type of amino acid residue, may yield enzymes with desired properties, thereby increasing their potential application. Ma and Nakai (1980) reported that chemical modification of carboxyl groups in porcine pepsin improved the stability of the enzyme near neutral pH. Yada and Nakai (1986a) proposed that the high MC/ PA of chymosin may be related to its high hydrophobicity and low charge.…”

Chemical modification of chloroperoxidase for enhanced stability and activity

“…maleic or phthalic) has been used [11][12]. Modifications of carboxyl groups of the protein (Glu and Asp) commonly involve the activation of the carboxyl group by a water-soluble carbodiimide and the subsequent reaction of the activated carboxyl group with a nucleophile [13][14].…”