Spectroscopic studies of the interfacial binding of Humicola lanuginosa lipase†

Abstract

The interaction of Humicola lanuginosa lipase (HLL) with small unilamellar vesicles of 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG) and in the presence of tributyrin (TB) as a substrate was studied by the use of steady-state fluorescence techniques. An inactive mutant with the serine from the catalytic triad changed by alanine (S146A) was used in experiments with TB to avoid interferences from product formation. HLL binds to POPG vesicles in an active or open form for the catalytic turnover, and therefore POPG provides a suitable system for studying the conformational changes involving the movement of the loop of amino acids that covers the active site of the enzyme in solution. Tryptophan (Trp) fluorescence shows that HLL binding to POPG occurs with a change in the environment of Trp residue(s) and that there is only one type of bound form, even in the presence of TB. Accessibility to aqueous quenchers indicates shielding of Trp in the membrane. Fluorescence anisotropy of the enzyme increases on binding to the vesicles, indicating restricted rotational freedom for the Trp due to penetration in the bilayer. Resonance energy transfer experiments using an interfacial membrane probe, 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene p-toluenesulfonate (TMA-DPH), and an internal membrane probe, 1,6-diphenylhexa-1,3,5-triene (DPH), indicate that HLL does not penetrate very deeply in the hydrophobic core of the membrane, but preferentially stays close to the lipid interface. Addition of substrate (TB) does not result in any additional changes in the spectroscopic properties of HLL. It is suggested that the observed changes are due to the ‘opening of the lid’ on binding to POPG vesicles, leaving the active site accessible for the substrate to bind.