McGeehan G M, Bickett D M, Green M, Kassel D, Wiseman J S, Berman J
Glaxo Inc. Research Institute, Research Triangle Park, North Carolina 27709.
J Biol Chem. 1994 Dec 30;269(52):32814-20.
The peptide substrate specificities of two matrix metalloproteinases (MMPs), interstitial collagenase (MMP-1), and 92-kDa gelatinase (MMP-9), have been examined. Starting with the parent substrate, Dnp-Pro-Leu-Gly approximately Leu-Trp-Ala-D-Arg-NH2, four separate substrate mixtures were synthesized at subsites P2(Leu) through P2'(Trp). These mixtures contained either naturally occurring L-amino acids, D-amino acids, or either of two distinct sets of miscellaneous amino acids. Combined, these mixtures gave 88 unique substitutions at each position and, over the four subsites, represented 352 potential substrates. Optimal substrates were identified using a combined high performance liquid chromatography/mass spectrometry analysis as previously reported. The results gave an extended profile of the substrate specificities for both MMP-1 and MMP-9 at subsites P2(Leu) through P2'(Trp). Using the data obtained from the mapping, a new peptide substrate, Dnp-Pro-Cha-Abu approximately Smc-His-Ala-D-Arg-NH2 (where Dnp is 2,4-dinitrophenyl, Cha is cyclohexylalanine, Abu is alpha-aminobutyric acid, and Smc is S-methylcysteine) was designed and characterized. This peptide showed a 36-fold improvement in turnover (kcat/Km) versus the parent substrate by interstitial collagenase. In addition, some collagenase subsite specificities described here were found to be different from those previously reported. Experimental data show that the observed selectivity is dependent on the original peptide template employed, which has broader implications for substrate specificity studies.
已对两种基质金属蛋白酶（MMPs），即间质胶原酶（MMP-1）和92 kDa明胶酶（MMP-9）的肽底物特异性进行了研究。从亲本底物Dnp-Pro-Leu-Gly≈Leu-Trp-Ala-D-Arg-NH₂开始，在P2（Leu）至P2′（Trp）亚位点合成了四种单独的底物混合物。这些混合物包含天然存在的L-氨基酸、D-氨基酸或两组不同的杂类氨基酸中的任何一种。这些混合物组合起来，在每个位置产生了88种独特的取代，并且在四个亚位点上代表了352种潜在底物。如先前报道的那样，使用高效液相色谱/质谱联用分析鉴定了最佳底物。结果给出了MMP-1和MMP-9在P2（Leu）至P2′（Trp）亚位点的底物特异性扩展图谱。利用从图谱获得的数据，设计并表征了一种新的肽底物Dnp-Pro-Cha-Abu≈Smc-His-Ala-D-Arg-NH₂（其中Dnp是2,4-二硝基苯基，Cha是环己基丙氨酸，Abu是α-氨基丁酸，Smc是S-甲基半胱氨酸）。与间质胶原酶的亲本底物相比，该肽的周转（kcat/Km）提高了36倍。此外，此处描述的一些胶原酶亚位点特异性与先前报道的不同。实验数据表明，观察到的选择性取决于所使用的原始肽模板，这对底物特异性研究具有更广泛的意义。
