Department of Biochemistry and Molecular Biology, University of Parma, Italy.
Arch Biochem Biophys. 2011 Jan 15;505(2):178-85. doi: 10.1016/j.abb.2010.10.005. Epub 2010 Oct 16.
The pyridoxal 5'-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase (OASS) catalyzes the synthesis of cysteine in bacteria and plants. In bacteria two isoenzymes are present, OASS-A and OASS-B, with distinct structural, functional, and regulatory properties. In order to gain a deeper insight into OASS-B dynamic and functional properties, single and double mutants of the three tryptophan residues, Trp28, Trp159, and Trp212, were prepared and their fluorescence emission properties were characterized in the absence and presence of substrate and ligands by steady-state and time-resolved spectrofluorimetry. Residue Trp28 was found to be mainly responsible for Trp fluorescence emission, whereas Trp212, located in a highly flexible region near the active site, is mainly responsible for an energy-transfer to PLP leading to an emission at 500 nm. Not surprisingly, mutation of Trp212 affects OASS-B activity. Trp159 slightly contributes to both direct emission and energy transfer to PLP. Time-resolved fluorescence measurements confirmed these findings, observing a third longer tryptophan lifetime for apo-OASS-B, in addition to the two lifetimes that are present in the holo-enzyme and mutants. A comparison with the emissions previously determined for OASS-A indicates that OASS-B active site is likely to be more polar and flexible, in agreement with a broader substrate specificity and higher catalytic efficiency.
磷酸吡哆醛（PLP）依赖性酶 O-乙酰丝氨酸硫羟化酶（OASS）在细菌和植物中催化半胱氨酸的合成。在细菌中存在两种同工酶，OASS-A 和 OASS-B，具有不同的结构、功能和调节特性。为了更深入地了解 OASS-B 的动态和功能特性，制备了三个色氨酸残基（Trp28、Trp159 和 Trp212）的单突变体和双突变体，并通过稳态和时间分辨荧光光谱法在没有和存在底物和配体的情况下表征了它们的荧光发射特性。发现残基 Trp28 主要负责色氨酸荧光发射，而位于靠近活性位点的高度灵活区域的 Trp212 主要负责将能量转移到 PLP 上，导致在 500nm 处发射。毫不奇怪，Trp212 的突变会影响 OASS-B 的活性。Trp159 对直接发射和向 PLP 的能量转移都有轻微的贡献。时间分辨荧光测量证实了这些发现，除了在全酶和突变体中存在的两个寿命外，apo-OASS-B 还观察到第三个更长的色氨酸寿命。与先前为 OASS-A 确定的发射相比，这表明 OASS-B 的活性位点可能更具极性和灵活性，这与更广泛的底物特异性和更高的催化效率一致。
