Studies of a phospholipase B from Penicillium notatum substrate specificity and properties of active site

Abstract

1. 1. Penicillium notatum phospholipase B catalyzes the hydrolysis of the 1- and 2-acyl ester bonds of 1,2-diacyl- sn-glycero-3-phosphocholine (phospholipase B activity), and of the 1-or 2-acyl ester bond of monoacyl- sn-glycero-3-phosphocholine (lysophospholipase activity), with concomitant release of fatty acid(s) and glycerophosphocholine. It also catalyzes the deacylation of the 3-acyl ester bond 2,3-diacyln- sn-glycero-1-phosphocholine but not the 2-acyl ester bond. 2. 2. Phospholipase A 1 and A 2 activities of the phospholipase B free from the following lysophospholipase activity could be determined using 2,3-dipalmitoylsn-glycero-1-phosphocholine and 1- O-alkyl-2-palmitoyl- sn-glycero-3-phosphocholine, respectively. 3. 3. Modification of phospholipase B by the action of an endogenous protease (s) reduced the phospholipase B activity (Okumura, T., Kimura, S. and Saito, K. (1980) Biochim. Biophys. Acta 617, 264–273). With reduction of the phospholipase B activity of the modified enzyme, its a 1 and A 2 activities were also reduced, but its lysophospholipase activity was almost the same as that of the native enzyme. 4. 4. V ratios of the native and modified phospholipase B towards 1,2-dioctanoyl-, 1,2-didecanoyl-, and 1,2-dipalmitoyl- sn-glycero-3-phosphocholines were 0.79, 0.35, and 0.21, respectively. Of these substrates, 1,2-dioctanoyl-snglycero-3-phosphocholine was used as large disc-shaped or elongated cylindrical micelles, and other substrates as sonicated vesicles in the system. The apparent K m values of both types of enzyme decreased with increase in the chain length of the constituent fatty acids of these substrates. 5. 5. Triton X-100 stimulated the phospholipase B activity of both the native and modified forms of enzyme. The V values of phospholipase B activity of the two forms towards 1,2-dipalmitoyl- sn-glycero-3-phosphocholine in the presence of Triton X-100 were almost the same. 6. 6. The effect of chloramphenicol on phospholipase B activity was stimulatory and that of chlorpromazine was biphasic, stimulatory or inhibitory, depending on its concentration. 7. 7. Modifications of the phospholipase B with N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and phenylglyoxal inhibited phospholipase B and lysophospholipase activities to similar extents.