1egq
From Proteopedia
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ENHANCEMENT OF ENZYME ACTIVITY THROUGH THREE-PHASE PARTITIONING: CRYSTAL STRUCTURE OF A MODIFIED SERINE PROTEINASE AT 1.5 A RESOLUTION
Overview
Three-phase partitioning is fast developing as a novel bioseparation, strategy with a wide range of applications including enzyme stability and, enhancement of its catalytic activity. Despite all this, the enzyme, behaviour in this process still remains unknown. A serine proteinase, proteinase K, was subjected to three-phase partitioning (TPP). A 3 ml, volume of proteinase K solution (3 mg/ml in 0.05 M acetate buffer, pH 6.0), was brought to 30% (w/v) ammonium sulphate saturation by addition of, saturated ammonium sulphate. tert-Butanol (6 ml) was added to this, solution and the mixture was incubated at 25 degrees C for 1 h. The, precipitated protein in the mid-layer was dissolved in 3 ml of 0.05 M, acetate buffer, pH 6.0. The specific activity of the processed enzyme was, estimated and was found to be 210% of the original enzyme activity. In, order to understand the basis of this remarkable enhancement of the enzyme, activity, the structure of the TPP-treated enzyme was determined by X-ray, diffraction at 1.5 A resolution. The overall structure of the TPP-treated, enzyme is similar to the original structure in an aqueous environment. The, hydrogen bonding system of the catalytic triad is intact. However, the, water structure in the substrate binding site has undergone a, rearrangement as some of the water molecules are either displaced or, completely absent. Two acetate ions were identified in the structure. One, is located in the active site and seems to mimic the role of water in the, enzyme activity and stability. The other is located at the surface of the, molecule and is involved in stabilizing the local structure of the enzyme., The most striking observation in respect of the present structure pertains, to a relatively higher overall temperature factor (B = 19.7 A(2)) than the, value of 9.3 A(2) in the original enzyme. As a result of a higher, B-factor, a number of residues, particularly their side chains, were found, to adopt more than one conformation. It appears that the protein exists in, an excited state which might be helping the enzyme to function more, rapidly than the original enzyme in aqueous media. Summarily, the basis of, increased enzymatic activity could be attributed to (i) the presence of an, acetate ion at the active site and (ii) its excited state as reflected by, an overall higher B-factor.
About this Structure
1EGQ is a Single protein structure of sequence from Engyodontium album with CA and ACY as ligands. Active as Peptidase K, with EC number 3.4.21.64 Full crystallographic information is available from OCA.
Reference
Enhancement of enzyme activity through three-phase partitioning: crystal structure of a modified serine proteinase at 1.5 A resolution., Singh RK, Gourinath S, Sharma S, Roy I, Gupta MN, Betzel C, Srinivasan A, Singh TP, Protein Eng. 2001 May;14(5):307-13. PMID:11438752
Page seeded by OCA on Tue Nov 20 13:59:36 2007
