Modulation of the catalytic mechanism of hen egg white lysozyme (HEWL) by photochromism of azobenzene

Hen egg white lysozyme (HEWL) (EC3.2.1.17) was modified with azobenzene (AZB) and the influences of photochromism of AZB on the enzyme activity was studied. Mass spectrometry (MALDI-TOF MS) study revealed that one molecule of AZB-4-carboxylic acid binds to ɛ-amino group of Lys33 located in the back space of the substrate binding site F, through amide bond. The reversible photochromism of AZB in HEWL was confirmed by spectrophotometric measurement. The irradiation with UV light to trans-AZB HEWL (AZBt-L) generated two types of cis-AZB HEWL (AZBc-L) which were named as AZBcl-L and AZBc2-L in elution order on a reversed phase HPLC. The fraction at the photostationary state (366 nm) was consisted of AZBt-L (20%), AZBcl-L (50%), and AZBc2-L (30%). Catalytic efficiencies (kcat/Km) of AZB-Ls lowered to one-half of the native enzyme efficiency. Maximum initial rate (Vmax) and Michaelis-Menten constant (Km) of AZBt-L were 1.50 × 10−1 mg ml−1 s−1, 3.00 × 10−1 mg ml−1, respectively. The fraction of AZBc-Ls showed 1.05 × 10−1 of Vmax and 1.82 × 10−1 of Km. The isomerization from trans-form to cis-form accompanied the decrease of Vmax and the increase of the affinity for substrate (Km). 1H NMR study suggests that the modulation may be caused by the configurational change of Glu35 via Trpl08 induced by the photochromism of AZB.