Wolz R L
Department of Biochemistry and Molecular Biology, Penn State College of Medicine, Milton S. Hershey Medical Center, Hershey 17033.
Arch Biochem Biophys. 1994 Apr;310(1):144-51. doi: 10.1006/abbi.1994.1150.
Astacin, a 23-kDa monomeric metalloprotease from the crayfish digestive tract, and meprin A, a 360-kDa tetrameric metalloprotease from the mouse kidney, are 30% identical in the amino acid sequence of their protease domains (Dumermuth et al., 1991, J. Biol. Chem. 266, 21381). The two were compared kinetically using a variety of substrates and inhibitors. Both enzymes degraded azocasein; meprin A had a twofold higher molar specific activity. Succinyl-Ala-Ala-Ala-p-nitroanilide was cleaved by both enzymes at the Ala-p-nitroanilide bond, indicating that in contrast to many metallopeptidases, peptidases of the astacin family are capable of arylamidolysis. Several peptides from a series of chromogenic and fluorogenic bradykinin analogs, originally designed for mapping the active site of meprin A, were found to be excellent substrates for astacin. Both enzymes cleaved most substrates at the site corresponding to the Phe5-Ser6 bond in native bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg). The peptide 2ABz-Arg-Pro-Ile-Phe decreases Ser-Pro-Phe(4-nitro)-Arg was found to have the highest kcat/Km ratio for both peptidases (4.5 x 10(5) M-1 s-1 for meprin A and 2.7 x 10(6) M-1 s-1 for astacin). The two enzymes were found to have several other substrates in common, indicating that some of these peptides could be substrates for the other putative proteases in the astacin family. Acetyl-Arg-Pro-Gly-Tyr-NHOH, an inhibitor which likely binds to the S subsites, was found to act on both enzymes via a predominantly non-competitive mechanism, whereas NHOH-succinyl-Pro-Phe-Arg, which likely binds to the S' subsites, was competitive. Significant specificity differences between astacin and meprin A were seen in substrates and inhibitors with bulky groups in the P1' position. Substrates with Arg, Lys, or Phe in the P1' position were cleaved 10(3)-10(4) times faster by meprin A than by astacin. Actinonin, a naturally occurring peptide hydroxamate with a pentyl group in P1', was a very potent competitive inhibitor of meprin A (Ki = 1.35 x 10(-7) M), but had a 1000-fold weaker affinity for inhibition of astacin. These kinetic differences indicate that the S1' subsite is smaller in astacin than in meprin A and correlate with structural differences seen in the three-dimensional models of the two enzymes.
虾红素是一种来自小龙虾消化道的23千道尔顿单体金属蛋白酶，而膜型金属蛋白酶A是一种来自小鼠肾脏的360千道尔顿四聚体金属蛋白酶，它们的蛋白酶结构域氨基酸序列有30%的同源性（杜默穆特等人，1991年，《生物化学杂志》266卷，21381页）。使用多种底物和抑制剂对这两种酶进行了动力学比较。两种酶都能降解偶氮酪蛋白；膜型金属蛋白酶A的摩尔比活性高两倍。琥珀酰 - 丙氨酸 - 丙氨酸 - 丙氨酸 - 对硝基苯胺在丙氨酸 - 对硝基苯胺键处被两种酶切割，这表明与许多金属肽酶不同，虾红素家族的肽酶能够进行芳基酰胺水解。最初设计用于绘制膜型金属蛋白酶A活性位点的一系列显色和荧光缓激肽类似物中的几种肽，被发现是虾红素的优良底物。两种酶都在天然缓激肽（精氨酸 - 脯氨酸 - 脯氨酸 - 甘氨酸 - 苯丙氨酸 - 丝氨酸 - 脯氨酸 - 苯丙氨酸 - 精氨酸）中对应苯丙氨酸5 - 丝氨酸6键的位点切割大多数底物。肽2ABz - 精氨酸 - 脯氨酸 - 异亮氨酸 - 苯丙氨酸 - 丝氨酸 - 脯氨酸 - 苯丙氨酸（4 - 硝基） - 精氨酸被发现对两种肽酶具有最高的催化常数与米氏常数比值（膜型金属蛋白酶A为4.5×10⁵ M⁻¹ s⁻¹，虾红素为2.7×10⁶ M⁻¹ s⁻¹）。发现这两种酶还有其他几种共同底物，这表明其中一些肽可能是虾红素家族中其他假定蛋白酶的底物。乙酰 - 精氨酸 - 脯氨酸 - 甘氨酸 - 酪氨酸 - NHOH是一种可能与S亚位点结合的抑制剂，被发现通过主要是非竞争性机制作用于两种酶，而NHOH - 琥珀酰 - 脯氨酸 - 苯丙氨酸 - 精氨酸可能与S'亚位点结合，是竞争性的。在P1'位置有庞大基团的底物和抑制剂中，虾红素和膜型金属蛋白酶A之间存在显著的特异性差异。P1'位置含有精氨酸、赖氨酸或苯丙氨酸的底物被膜型金属蛋白酶A切割的速度比被虾红素快10³ - 10⁴倍。放线菌素是一种在P1'位置带有戊基的天然存在的肽羟肟酸，是膜型金属蛋白酶A的一种非常有效的竞争性抑制剂（抑制常数Ki = 1.35×10⁻⁷ M），但对虾红素的抑制亲和力弱1000倍。这些动力学差异表明，虾红素中的S1'亚位点比膜型金属蛋白酶A中的小，并且与两种酶的三维模型中观察到的结构差异相关。
