Both the phosphatidylinositol-hydrolysing and the phosphatidylcholine-hydrolysing phospholipases C have been implicated in the generation of second messengers in mammalian cells. The phosphatidylcholine-hydrolysing phospholipase C (PLC) from Bacillus cereus, a monomeric protein containing 245 amino-acid residues, is similar to some of the corresponding mammalian proteins. This, together with the fact that the bacterial enzyme can mimic the action of mammalian PLC in causing, for example, enhanced prostaglandin biosynthesis, suggests that B. cereus PLC can be used as a model for the hitherto poorly characterized mammalian PLCs. We report here the three-dimensional structure of B. cereus PLC at 1.5 A resolution. The enzyme is an all-helix protein belonging to a novel structural class and contains, at least in the crystalline state, three Zn2+ in the active site. We also present preliminary results from a study at 1.9 A resolution of the complex between PLC and inorganic phosphate (Pi) which indicate that the substrate binds directly to the metal ions.

No abstract

No abstract

The inactivation of glyceraldehyde-3-phosphate dehydrogenase by hydrogen peroxide has (a) I n the absence of catalysts been investigated. The reaction obeyed two rate equations : did not oxidize the "essential" sulphydryl groups of the enzyme to disulphide, but to sulphenic acid residues. Peroxide-inactivated glyceraldehyde-3-phosphate dehydrogenase could be fully reactivated and sulphydryl oxidation fully reversed if the enzyme was treated immediately with excess small thiol. The reversibility of inactivation decreased progressively if thiol treatment was delayed. 4 hours after peroxide treatment, neither enzyme inactivation nor sulphydryl oxidation could be reversed.Although SH-enzymes are normally inactivated by most oxidizing agents [l], relatively little information is available about the precise mechanisms involved. Thus, the powerful oxidizing agent, H,O,, inactivates most SH-enzymes [l] and in the case of papain, the inactivation is caused exclusively by SHoxidation [Z]. The nature of the products formed after H,O,-oxidation of protein thiols is not clear, although they are frequently assumed to be disulphides.The purpose of the present work was to study the mechanisms of inactivation of SH-enzymes by peroxides. For this purpose glyceraldehyde-3-phosphate dehydrogenase was chosen. This enzyme is inactivated by SH-reagents such as mercurials and alkylating activation of glyceraldehyde-3-phosphate dehydrogenase has been published. It would therefore seem that this enzyme is a very suitable one to use for a study of the mechanism of peroxide-inactivation of SH-enzymes. Moreover, our previous work indicated that whereas o-iodosobenzoate caused disulphide formation in the protein monothiol, yeast cytochrome c , peroxides did not [7]. As there is good evidence that o-iodosobenzoate causes intramolecular disulphide formation in glyceraldehyde-3-phosphate dehydrogenase [4] this enzyme is also very suitable for testing out the previous suggestion [7] that peroxide-oxidation of protein thiols rarely produces disulphides. MATERIALS AND METHODS MaterialsNAD, o-iodosobenzoate, glutathione, cysteine, 5,5'-dithiobis(2-nitrobenzoic acid), and horse heart cytochrome c, TypeII, were obtained from Sigma Chemical Co. (St. Louis, Mo.). Sodium borohydride