Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions

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Title: Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions
Authors: Ceruso, Mariangela
Howe, Nicole
Malthouse, J.Paul G.
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Date: Feb-2012
Online since: 2012-01-30T12:51:57Z
Abstract: The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82 ppm and 182.87 on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5 ppm to 182.9 ppm on binding to thermolysin. From pH studies we conclude that the pKa of the inhibitor carboxylate group is lowered by at least 1.5 pKa units when it binds to either enzyme. The signal at ~183 ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of carboxylate carbon of Z-[1-13C]-L-tryptophan is ≤ 3.71 Å from the active site cobalt atom of thermolysin. We conclude that the side chain of Z-[1-13C]-L-tryptophan is not bound in the S2' subsite of thermolysin. As the chemical shifts of the carboxylate carbons of the bound inhibitors are all ~183 ppm we conclude that they are all bound in a similar way most probably with the inhibitor carboxylate group directly coordinated to the active site zinc atom. Our spectrophotometric results confirm that the active site zinc atom is tetrahedrally coordinated when the inhibitors Z-tryptophan or Z-phenylalanine are bound to thermolysin.
Funding Details: Science Foundation Ireland
Higher Education Authority
Other funder
Type of material: Journal Article
Publisher: Elsevier B.V.
Journal: Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics
Volume: 1824
Issue: 2
Start page: 303
End page: 310
Copyright (published version): 2011 Elsevier B.V.
Keywords: ThermolysinStromelysinNMRAmino acidsBindingCobalt
Subject LCSH: Metalloproteinases
Amino acids
Nuclear magnetic resonance
DOI: 10.1016/j.bbapap.2011.10.007
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Language: en
Status of Item: Peer reviewed
Appears in Collections:Biomolecular and Biomedical Science Research Collection

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