Understanding the P1' specificity of the matrix metalloproteinases: effect of S1' pocket mutations in matrilysin and stromelysin-1.
Understanding the P1' specificity of the matrix metalloproteinases: effect of S1' pocket mutations in matrilysin and stromelysin-1.
复制标题
了解基质金属蛋白酶的 P1 特异性：Matrilysin 和 Stromelysin-1 中 S1 口袋突变的影响。
DOI：
10.1021/bi9601969
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
VanWart,HE
中科院分区：
文献类型：
--
作者：
Welch,AR;Holman,CM;Huber,M;Brenner,MC;Browner,MF;VanWart,HE
基质溶素（MAT）比在肽和蛋白质底物的P1'位置具有芳香族侧链的残基更喜欢亮氨酸，而基质溶素（HFS）具有更广泛的特异性。这些酶的X射线结构表明，它们各自的S1'亚位点的不同主要是由于214和215位上存在的氨基酸。为了检测这些残基在决定P1'特异性中所起的作用，基质溶解素中这些位置的氨基酸被基质溶解素中发现的氨基酸所取代（MAT：Y214 L，MAT：A215 V，和MAT：Y214 L/A215 V）。MAT：A215 V的特异性和活性与野生型基质溶解素相似。然而，MAT：Y214 L和MAT：Y214 L/A215 V两者都具有P1'特异性，其与基质溶解素相比更类似于基质溶解素。具体地说，相对于野生型基质溶解素，这些酶对在亚位点P1'具有Leu的肽底物表现出8- 9倍的降低inkcat/Km。这主要是由于inkcat减少了约5倍。Km值仅部分增加到观察到的溶基质素的值。对野生型和突变型基质溶解素与P1'位置具有Leu和Tyr残基的底物的稳态前反应的研究证实，这些反应的Km值反映了底物结合的KD值。因此，用亮氨酸替换基质溶素S1'口袋中的单个酪氨酸残基改变了其P1'特异性，使其类似于基质溶素的特异性。相比之下，将基质溶解素（HFS：L214 Y/V215 A）的S1'亚位点改变为类似于基质溶解素增加了活性（即，较高的kcat/KM），对亮氨酸和在P1'位置具有芳香族侧链的残基的肽底物的特异性仅部分增加。这些活性的增加是这些反应的Km值降低的结果。
Matrilysin (MAT) prefers leucine over residues that have aromatic side chains at the P1‘ position of peptide and protein substrates, while stromelysin (HFS) has a broader specificity. The X-ray structures of these enzymes show that their respective S1‘ subsites differ primarily due to the amino acids present at positions 214 and 215. To examine the role that these residues play in determining P1‘ specificity, the amino acids at these positions in matrilysin have been replaced by those found in stromelysin (MAT:Y214L, MAT:A215V, and MAT:Y214L/A215V). The specificity and activity of MAT:A215V are similar to those of wild type matrilysin. Both MAT:Y214L and MAT:Y214L/A215V, however, have P1‘ specificities that are more similar to stromelysin than matrilysin. Specifically, these enzymes exhibit an 8- to 9-fold reduction inkcat/KMtoward a peptide substrate with Leu in subsite P1‘ relative to wild type matrilysin. This is predominantly the result of an approximate 5-fold decrease inkcat. TheKMvalues only partially increase toward the value observed for stromelysin. Studies of the pre-steady-state reaction of wild type and mutant matrilysin with substrates with Leu and Tyr residues in the P1‘ position confirm that theKMvalues for these reactions reflectKDvalues for substrate binding. Thus, replacement of a single tyrosine residue in the S1‘ pocket of matrilysin by leucine alters its P1‘ specificity to resemble that of stromelysin. In contrast, alteration of the S1‘ subsite of stromelysin (HFS:L214Y/V215A) to resemble matrilysin increases activity (i.e., higherkcat/KM) toward peptide substrates with both leucine and residues with aromatic side chains in the P1‘ position with only a partial increase in specificity for Leu. These increases in activity are the result of decreases in theKMvalues for these reactions.