Department of Infectious Disease, Imperial College London, London, W12 0NN, UK.
Centre for Bacterial Resistance Biology, Imperial College London, London, SW7 2AZ, UK.
Sci Rep. 2023 Nov 3;13(1):19052. doi: 10.1038/s41598-023-46036-9.
The Streptococcus pyogenes cell envelope protease (SpyCEP) is vital to streptococcal pathogenesis and disease progression. Despite its strong association with invasive disease, little is known about enzymatic function beyond the ELR CXC chemokine substrate range. As a serine protease, SpyCEP has a catalytic triad consisting of aspartate (D151), histidine (H279), and serine (S617) residues which are all thought to be mandatory for full activity. We utilised a range of SpyCEP constructs to investigate the protein domains and catalytic residues necessary for enzyme function. We designed a high-throughput mass spectrometry assay to measure CXCL8 cleavage and applied this for the first time to study the enzyme kinetics of SpyCEP. Results revealed a remarkably low Michaelis-Menton constant (K) of 82 nM and a turnover of 1.65 molecules per second. We found that an N-terminally-truncated SpyCEP C-terminal construct containing just the catalytic dyad of H279 and S617 was capable of cleaving CXCL8 with a similar K of 55 nM, albeit with a reduced substrate turnover of 2.7 molecules per hour, representing a 2200-fold reduction in activity. We conclude that the SpyCEP C-terminus plays a key role in high affinity substrate recognition and binding, but that the N-terminus is required for full catalytic activity.
化脓性链球菌细胞包膜蛋白酶(SpyCEP)对链球菌的发病机制和疾病进展至关重要。尽管它与侵袭性疾病密切相关,但除了 ELR CXC 趋化因子底物范围之外,对其酶功能知之甚少。作为一种丝氨酸蛋白酶,SpyCEP 的催化三联体由天冬氨酸(D151)、组氨酸(H279)和丝氨酸(S617)残基组成,这些残基被认为对完全活性是必需的。我们利用一系列 SpyCEP 构建体来研究酶功能所需的蛋白质结构域和催化残基。我们设计了一种高通量质谱测定法来测量 CXCL8 的切割,并首次将其应用于研究 SpyCEP 的酶动力学。结果显示出非常低的米氏常数(K)为 82 nM,周转率为每秒 1.65 个分子。我们发现,仅包含催化二联体 H279 和 S617 的 N 端截断 SpyCEP C 端构建体能够以类似的 K 55 nM 切割 CXCL8,尽管底物周转率降低至每小时 2.7 个分子,活性降低了 2200 倍。我们得出结论,SpyCEP C 端在高亲和力底物识别和结合中起关键作用,但 N 端对于完全的催化活性是必需的。