Structural characterization of two artificial enzymes based on adipocyte lipid binding protein

J. J. Ory, H. Kuang, R. Davies, A. Mazhary, Mark D Distefano, L. J. Banaszak

Research output: Contribution to journalArticlepeer-review

Abstract

Adipocyte Lipid Binding Protein (ALBP) is a small (14.5 kI)a) ten stranded .#barrel protein found in nlammalian fat cells. Tile crystal structures of various ht)lo-fornls of ALBP have been solved, and show the fatty acid ligand bound in a large (approx. 600 A3) cavity isolated from bulk solvent. On examination of t h(, cavity, it al/)peared to be a good site for the creation of an artificial enzyme, as numerous well-defined crystal structures of ALBP were available, and past studies have shown lhe conformation to be reasonably tolerant to mntagenest. The first stage was to test whether catalysis included enantioselectivity (Kuanget. al.. 1996). With thisgoalin mind. ALBP was modified at ('11-. which points into the binding cavity, so it contained either a phenanthroline ( :\ Pttt';N ) or pyri doxami ne( 3, P X ) group. A P X catalyzes the transamination of (-k(qo to (-amino acids, yielding up to 94% ee depending on initial substrate. APtlEN-('u++ catalyzes the hydrolysis of ainino acid esters to amino acids, with ('s ranging from 39% to 86%. The crystal structures of both forms have been solved to good Rfacto/Rfree (APHEN - 18.8%/27.2%, APX - 19.8/29.1. l"rom these structures, it is apparent that the adducts reduce the internal cavity volume, sterically liIniting substrate interactions with the reactive groups. Also. the (avity of ALBP contains charged groups which could orient bound substrates. Both x ray structures as well as potential intermediate structures will be presonted, and an analysis of the structural basis for onantiosoloivity will be made. Supported by Nltt GM 13925.

Original languageEnglish (US)
JournalFASEB Journal
Volume11
Issue number9
StatePublished - Dec 1 1997

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