Side chain interactions at the dimer interface of Vibrio alkaline phosphatase determine its reactivity, stability and desthiobiotin inhibition

Vibrio alkaline phosphatase (VAP) is a homodimeric, psychrophilic enzyme. A homodimer is, by far, the most dominant quaternary enzyme structure found in nature. It is therefore obvious that this particular structure gives the enzymes some advantage in the evolutionary race. Many suggestions have been proposed, from stabilization effects due to a smaller surface to volume ratio, an increased ratio of active sites to surface and more recently, the idea of “cross-talking” between subunits where only one active site has catalytic capabilities at any given time. In this study, the enzyme was mutated to try and disrupt the dimer equilibrium. A few methods are being developed to assess this equilibrium and three of them were used for this study. CD spectroscopy monitors a change in the enzymes circular dichroism due to changes in its secondary structure. Fluorescence spectroscopy detects a change in the environment of Trp residues that cause a shift in λmax when the protein is denatured. Dilution induced dimer dissociation to monomers on the grounds of Le Chatelier’s principle. Previous studies have shown that VAP unfolding involves at least two intermediate states, one with an inactive dimer and one with unfolded monomers, before the enzyme is completely denatured, N2 → I2 → 2I → 2D. This study also assessed a possible inhibition of desthiobiotin (DDB) on VAP. DDB is used to elute purified enzyme samples of Strep-Tactin® affinity columns. Recently, crystal images of VAP in a solution containing DDB revealed some kind of interaction between the molecule and residues close to the active site. Results revealed inhibitory effect (Ki=0.091) at 0.25 mM DDB a 10-fold dilution compared to the concentration used for elution of purified enzymes. The exact characteristics of this inhibition are still unclear, but it is likely of a mixed noncompetitive nature.

Author dc:creator

• Sigríður Stefanía Hlynsdóttir 1991-

Contributors dc:contributor

• Háskóli Íslands

• Lífefnafræði
• Ensím

Language dc:language.iso

en

Handle dc:identifier.uri

http://hdl.handle.net/1946/30544

OAI identifier oai:identifier

oai:skemman.is:1946/30544