Marokházi Judit, Mihala Nikolett, Hudecz Ferenc, Fodor András, Gráf László, Venekei István
Department of Biochemistry, Eötvös Loránd University, Budapest, Hungary.
FEBS J. 2007 Apr;274(8):1946-56. doi: 10.1111/j.1742-4658.2007.05739.x. Epub 2007 Mar 12.
The aim of this study was the development of a sensitive and specific substrate for protease A (PrtA), a serralysin-like metzincin from the entomopathogenic microorganism, Photorhabdus. First, cleavage of three biological peptides, the A and B chains of insulin and beta-lipotropin, and of 15 synthetic peptides, was investigated. In the biological peptides, a preference for the hydrophobic residues Ala, Leu and Val was observed at three substrate positions, P2, P1' and P2'. At these positions in the synthetic peptides the preferred residues were Val, Ala and Val, respectively. They contributed to the efficiency of hydrolysis in the order P1' > P2 > P2'. Six amino acids of the synthetic peptides were sufficient to reach the maximum rate of hydrolysis, in accordance with the ability of PrtA to cleave three amino acids from both the N- and the C-terminus of some fragments of biological peptides. Using the best synthetic peptide, a fluorescence-quenched substrate, N-(4-[4'(dimethylamino)phenylazo]benzoyl-EVYAVES-5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid, was prepared. The approximately 4 x 10(6) M(-1) x s(-1) specificity constant of PrtA (at K(m) approximately 5 x 10(-5) M and k(cat) approximately 2 x 10(2) s(-1)) on this substrate was the highest activity for a serralysin-type enzyme, allowing precise measurement of the effects of several inhibitors and pH on PrtA activity. These showed the characteristics of a metalloenzyme and a wide range of optimum pH, similar to other serralysins. PrtA activity could be measured in biological samples (Photorhabdus-infected insect larvae) without interference from other enzymes, which indicates that substrate selectivity is high towards PrtA. The substrate sensitivity allowed early (14 h post infection) detection of PrtA, which might indicate PrtA's participation in the establishment of infection and not only, as it has been supposed, in bioconversion.
本研究的目的是开发一种灵敏且特异的蛋白酶A（PrtA）底物，PrtA是一种来自昆虫病原微生物发光杆菌（Photorhabdus）的类解锯脂素金属锌蛋白酶。首先，研究了三种生物肽（胰岛素A链和B链以及β-促脂素）和15种合成肽的裂解情况。在生物肽中，在三个底物位置P2、P1'和P2'观察到对疏水残基丙氨酸（Ala）、亮氨酸（Leu）和缬氨酸（Val）的偏好。在合成肽的这些位置，优选的残基分别是缬氨酸、丙氨酸和缬氨酸。它们对水解效率的贡献顺序为P1' > P2 > P2'。合成肽的六个氨基酸足以达到最大水解速率，这与PrtA从一些生物肽片段的N端和C端切割三个氨基酸的能力一致。使用最佳合成肽制备了一种荧光猝灭底物N-(4-[4'(二甲氨基)苯基偶氮]苯甲酰基-EVYAVES-5-[(2-氨基乙基)氨基]萘-1-磺酸。PrtA对该底物的特异性常数约为4×10(6) M(-1)×s(-1)（K(m)约为5×10(-5) M，k(cat)约为2×10(2) s(-1)），是类解锯脂素型酶的最高活性，可精确测量几种抑制剂和pH对PrtA活性的影响。这些结果显示了其作为金属酶的特性以及与其他解锯脂素相似的较宽的最适pH范围。PrtA活性可以在生物样品（受发光杆菌感染的昆虫幼虫）中进行测量，而不受其他酶的干扰，这表明该底物对PrtA具有高选择性。底物的敏感性使得能够早期（感染后14小时）检测到PrtA，这可能表明PrtA不仅如人们所认为的那样参与生物转化，还参与感染的建立。
