Jaron Shulamit, Mains Richard E, Eipper Betty A, Blackburn Ninian J
Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering, Oregon Health & Science University, Beaverton, Oregon 97006-8921, USA.
Biochemistry. 2002 Nov 5;41(44):13274-82. doi: 10.1021/bi020404z.
The spectroscopic characterization of the H172A mutant of peptidylglycine alpha-hydroxylating monooxygenase (PHM) was undertaken to determine the importance of this Cu(H) ligand in the catalytic mechanism of PHM. Mutation of this histidine reduced the activity of the enzyme over 300-fold with little effect on the structure of the oxidized form. However, the reduced enzyme showed a decrease in the average Cu-N(His) distances from 1.96 A in wild-type PHM to 1.89 A in H172A associated with a change in the structure of Cu(H) from distorted T-shaped planar in the wild type to 2-coordinate in the mutant. Binding of CO was retained at the Cu(M) site (similar to wild type), and peptide substrate binding continued to activate a second site for CO binding. Confirmation of this substrate-induced CO binding site at Cu(H) was obtained through the observation that loss of the H172 Cu(H) ligand caused a 3 cm(-)(1) blue shift in the nu(CO) for this copper carbonyl. Possible mechanistic roles for the H172 ligand are discussed.
对肽基甘氨酸α-羟化单加氧酶（PHM）的H172A突变体进行了光谱表征，以确定该铜配体在PHM催化机制中的重要性。该组氨酸的突变使酶活性降低了300多倍，而对氧化形式的结构影响很小。然而，还原态的酶显示平均Cu-N（His）距离从野生型PHM中的1.96 Å减小到H172A中的1.89 Å，这与Cu（H）结构从野生型中的扭曲T形平面变为突变体中的二配位有关。CO在Cu（M）位点的结合得以保留（与野生型相似），并且肽底物结合继续激活第二个CO结合位点。通过观察到H172 Cu（H）配体的缺失导致该羰基铜的ν（CO）发生3 cm⁻¹的蓝移，证实了在Cu（H）处存在这种底物诱导的CO结合位点。讨论了H172配体可能的机制作用。
