The three-dimensional structure of the lipase-procolipase complex, co-crystallized with mixed micelles of phosphatidylcholine and bile salt, has been determined at 3 A resolution by X-ray crystallography. The lid, a surface helix covering the catalytic triad of lipase, adopts a totally different conformation which allows phospholipid to bind to the enzyme's active site. The open lid is an essential component of the active site and interacts with procolipase. Together they form the lipid-water interface binding site. This reorganization of the lid structure provokes a second drastic conformational change in an active site loop, which in its turn creates the oxyanion hole (induced fit).

Limited action of papain on the native forms of two cellobiohydrolases (CBH) from Trichoderma reesei (CBH I, 65 kDa, and CBH II,58 kDa) leads to the isolation of the respective core fragments (56 kDa and 45 kDa) which are fully active on small, soluble substrates, but have a strongly reduced activity (respectively 10% and 50% of the initial value) on microcrystalline cellulose (Avicel).By partial sequencing at the C terminus of the CBH I core and at the N terminus of the CBH I1 core the papain cleavage sites have been assigned in the primary structures (at about residue 431 and 82 respectively). This limited action of papain on the native enzymes indicates the presence of hinge regions linking the core to these terminal glycopeptides. The latter conserved sequences appear either at the C or N terminus of several cellulolytic enzymes from ~r~c~o d e r m a reesei [Teeri et al. (1987) Gene 51,43 -521. The specific activities of the intact enzymes and their cores on two forms of insoluble cellulose (crystalline, amorphous) differentiate the CBH I and CBH TI in terms of adsorption and catalytic properties, Distinct functions can be attributed to the terminal peptides: for intact CBH I1 the N-terminal region contributes in the binding onto both cellulose types; the homologous C-terminal peptide in CBH I, however, only affects the interaction with microcrystalline cellulose. It could be inferred that CBH I and its core bind preferentially to crystalline regions. This seems to be corroborated by the results of CBH I/CBH I1 synergism experiments.The filamentous fungus Trichoderma reesei presents an ideal system for the study of cellulose hydrolysis since it produces a variety of enzymes including endocellulase and exocellulase (cellobiohydrolases) and b-glucosidases (cellobiases) [l]. The primary structure of several of these proteins is presently known [2 -61 but no structure/function relationships have yet been established. The two cellobiohydrolases, CBH I and CBH I1 [7 -111, lack any apparent homology in their amino acid sequence except for a conserved region present at their C and N termini respectively [4].Their action is similar both with regard to their strong adsorption onto insoluble substrates and lack of carboxymethylcellulase activity. However, studies both with microcrystalline cellulose [9] and small, soluble substrates [I 21 reveal some differences in specificity. In addition, the two cellobiohydrolases interact synergistically both with each other and with endoglucanases on microcrystalline substrates In order to differentiate these enzymes further by their function and to investigate possible domain structure, proteolysis studies were performed. Preliminary results on the limited action of papain on native CBH I have been reported by us [16]. Two fragments were isolated: first, a heavily glycosylated peptide, tentatively identified with the C terminus; secondly, a core protein containing the active (hydrolytic) site. The terminal peptide was characterised as an independent structural and functional dom...

The DEAH/RNA helicase A (RHA) helicase family comprises proteins involved in splicing, ribosome biogenesis and transcription regulation. We report the structure of yeast Prp43p, a DEAH/RHA helicase remarkable in that it functions in both splicing and ribosome biogenesis. Prp43p displays a novel structural architecture with an unforeseen homology with the Ski2-like Hel308 DNA helicase. Together with the presence of a b-hairpin in the second RecA-like domain, Prp43p contains all the structural elements of a processive helicase. Moreover, our structure reveals that the C-terminal domain contains an oligonucleotide/oligosaccharide-binding (OB)-fold placed at the entrance of the putative nucleic acid cavity. Deletion or mutations of this domain decrease the affinity of Prp43p for RNA and severely reduce Prp43p ATPase activity in the presence of RNA. We also show that this domain constitutes the binding site for the G-patch-containing domain of Pfa1p. We propose that the C-terminal domain, specific to DEAH/RHA helicases, is a central player in the regulation of helicase activity by binding both RNA and G-patch domain proteins.