McDermott J R, Gibson A M, Oakley A E, Biggins J A
Medical Research Council, Neurochemical Pathology Unit, Newcastle General Hospital, England.
J Neurochem. 1991 May;56(5):1509-17. doi: 10.1111/j.1471-4159.1991.tb02045.x.
The main high molecular weight (650K) multicatalytic endopeptidase has been purified from postmortem human cerebral cortex. As in other tissues and species, this enzyme is composed of several subunits of 24-31K and has three distinct catalytic activities, as shown by the hydrolysis of the fluorogenic tripeptide substrates glutaryl-Gly-Gly-Phe-7-amido-4-methylcoumarin, benzyloxycarboxyl-Gly-Gly-Arg-7-amido-4-methylcoumarin, and benzyloxycarboxyl-Leu-Leu-Glu-2-naphthylamide with hydrophobic (Phe), basic (Arg), and acidic (Glu) residues in the P1 position, respectively. These activities are distinguishable by their differential sensitivity to peptidase inhibitors. The enzyme hydrolysed neuropeptides at pH 7.4 at multiple sites with widely differing rates, ranging from 113 nmol/min/mg for substance-P, down to 2 nmol/min/mg for bradykinin. The enzyme also had proteinase activity as shown by the hydrolysis of casein. For the hydrolysis of the Tyr5-Gly6 bond in luteinizing hormone-releasing hormone, the Km was 0.95 mM and the specificity constant (kcat/Km) was 4.7 X 10(3) M-1 s-1. The bond specificity of the enzyme at neutral pH was determined by identifying the degradation products of 15 naturally occurring peptide sequences. The bonds most susceptible to hydrolysis had a hydrophobic residue at P1 and either a small (e.g., -Gly or -NH2) or hydrophobic residue at P'1. Hydrolysis of -Glu-X bonds (most notably in neuropeptide Y) and the Arg6-Arg7 bond in dynorphin peptides was also seen. Thus the three activities identified with fluorogenic substrates appear to be expressed against oligopeptides.
主要的高分子量（650K）多催化内肽酶已从死后的人类大脑皮层中纯化出来。与其他组织和物种一样，这种酶由几个24 - 31K的亚基组成，并具有三种不同的催化活性，这通过荧光三肽底物戊二酰 - 甘氨酰 - 苯丙氨酰 - 7 - 氨基 - 4 - 甲基香豆素、苄氧羰基 - 甘氨酰 - 甘氨酰 - 精氨酰 - 7 - 氨基 - 4 - 甲基香豆素以及苄氧羰基 - 亮氨酰 - 亮氨酰 - 谷氨酰 - 2 - 萘酰胺的水解得以证明，它们在P1位置分别具有疏水（苯丙氨酸）、碱性（精氨酸）和酸性（谷氨酸）残基。这些活性可通过它们对肽酶抑制剂的不同敏感性来区分。该酶在pH 7.4时能在多个位点水解神经肽，速率差异很大，从P物质的113 nmol/分钟/毫克到缓激肽的2 nmol/分钟/毫克。该酶还具有蛋白酶活性，如酪蛋白的水解所示。对于促黄体激素释放激素中Tyr5 - Gly6键的水解，Km为0.95 mM，特异性常数（kcat/Km）为4.7×10³ M⁻¹ s⁻¹。通过鉴定15种天然存在的肽序列的降解产物，确定了该酶在中性pH下的键特异性。最易被水解的键在P1处有一个疏水残基，在P'1处有一个小的（如 - 甘氨酸或 - NH₂）或疏水残基。还观察到了 - 谷氨酸 - X键（最显著的是在神经肽Y中）以及强啡肽肽中的Arg6 - Arg7键的水解。因此，用荧光底物鉴定的这三种活性似乎是针对寡肽表达的。
