Phospholipase A enzymes are divided into two basic groups, depending on which acyl moiety is hydrolysed. Phospholipases Ai comprise a large group of enzymes some of which may also degrade neutral lipids and, so, act as lipases. Usually the enzymes have a wide specificity and act well on lysophospholipids. Their function in most cases is obscure - except for the role of lipases with phos-pholipase Ai activity in lipoprotein metabolism (Section 3.5.2 and below).
The first example of a phospholipase Ai to be purified was from E. coli, which actually has two separate enzymes - a detergent-resistant enzyme in the outer membrane and a detergent-sensitive enzyme in the cytoplasmic membrane and soluble fractions.
In animals, phospholipase Ai is present in lyso-somes and has an appropriately low pH optimum (about 4.0). It does not need Ca2+ for activity but Ca2+ and charged amphipaths influence hydrolysis by altering the surface charge on the substrate micelle or membrane.
Also present in animals are two enzymes that, while preferentially hydrolysing triacylglycerols, will also hydrolyse the bond at position 1 of phos-pholipids. These are the extra-hepatic lipoprotein lipase (Section 3.5.2) and the hepatic lipase. The latter enzyme is activated by apolipoprotein E to hydrolyse phospholipids and by phospholipids to act as a lipase.
Phospholipases A2 are extremely widespread in Nature and their activity was noted first by Bokay over a hundred years ago. He studied the degradation of phosphatidylcholine by pancreatic secre tions and at the turn of the twentieth century cobra venom phospholipase activity was discovered. These two sources of phospholipase have proved very useful for purifying and studying the enzyme.
Phospholipase A2 enzymes can be divided into four main groups - based both on their properties and on sequence similarities (Table 7.3). Sufficient quantities of the phospholipases from pancreas and cobra venom (Group I) have been purified for detailed X-ray analyses to be made. Detailed reaction mechanisms have also been proposed (see Waite, 1996, in Further Reading).
Pancreatic phospholipases are synthesized as zymogens that are activated by the cleavage of a heptapeptide by trypsin. Cleavage of this peptide exposes a hydrophobic sequence, which then allows interaction of the enzyme with phospholipid substrates. The enzyme is very stable and its seven disulphide bonds no doubt play a key role here. Chemical modification and NMR studies have shown clearly that the catalytic and binding sites are distinct in both the pancreatic and snake venom phospholipases A2. In confirmation, it is known that the zymogen form of the pancreatic enzyme is also active even though its binding site is masked. Ca2+ is absolutely required for activity and seems to interact with both the phosphate and carbamyl groups of the ester undergoing hydrolysis.
The sequences of pancreatic phospholipase A2 and the enzymes from the venoms of Elapids (cobras, kraits and mambas) are closely related in contrast to the enzymes from vipers [e.g. Crotalus (rattlesnakes)], which are Group II enzymes (Table 7.3). Subtle modifications in the sequence of the Group I-II enzymes causes significant changes in their crystal structure and substrate interaction. For example, pancreatic enzymes (Group IB) crystallize as monomers, rattlesnake (Group IIA) as dimers and cobra phospholipases A2 (Group IA) as trimers.
Phospholipase A2 enzymes also have other important metabolic functions in addition to the overall destruction of phospholipids as catalysed by digestive pancreatic or venom enzymes. An enzyme in mitochondrial membranes seems to be intimately connected with the energy state of this organelle. Thus, the phospholipase is inactive in fully coupled mitochondria and only becomes
Table 7.3 Phospholipase A2 enzymes
Source Location Mass (kDa) Ca2+ needed?
A Cobras, kraits B Pancreas
A Rattlesnakes, vipers B Gaboon viper
Group III Bee, lizard
Kidney, macrophages, platelets
Others Myocardium Liver, macrophages
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