Biophysical investigation of recombinant K5 lyase: structural implications of substrate binding and processing.
K5 lyase of coliphage K5A degrades the K5 polysaccharide of encapsulated E. coli strains expressing the K5 antigen thereby contributing to virus binding and infection. We have investigated the affinities of the recombinant enzyme for different GAG ligands by isothermal fluorescence titrations and correlated them with substrate processing and protein structural changes. Chondroitin sulfate (CS) and heparan sulfate (HS) bound to K5 lyase with a Kd of 0.5 microM whereas heparin exhibited a Kd=1.1 microM. The natural substrate K5 polysaccharide displayed a similar apparent affinity as CS and HS but was the only ligand of the enzyme which induced a large structural rearrangement of the protein as detected by far-UV CD spectroscopy. Since significant enzymatic degradation was only found for the K5 polysaccharide peaking at 44 degrees C, but binding was also detected for heparin, we propose that the K5 lyase is able to discriminate between specific (acetylated/non-sulfated) and unspecific (acetylated/sulfated) ligands by its heparin binding motif in the C-terminus. This is proposed to be the origin for the enzyme's residual HS degrading activity.
Rek
Angelika
A
Institute of Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, University of Graz, Universitätsplatz 1, A-8010 Graz, Austria.
Thompson
James
J
Roberts
Ian S
IS
Kungl
Andreas J
AJ
D002809:Chondroitin Sulfates / Q000378:metabolism; D002942:Circular Dichroism; D004795:Enzyme Stability; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014466:Ultraviolet Rays
D002809:Chondroitin Sulfates; D029968:Escherichia coli Proteins; D006497:Heparan Sulfate; D011133:Polysaccharide-Lyases; D011994:Recombinant Proteins; C411971:KflA protein, E coli