SPECIFICITY DETERMINANTS OF ACYLAMINOACYL-PEPTIDE HYDROLASE
SPECIFICITY DETERMINANTS OF ACYLAMINOACYL-PEPTIDE HYDROLASE
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DOI：
10.1002/pro.5560010504
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发表时间：
1992-05-01
期刊：
影响因子：
8
通讯作者：
WOLD, F
中科院分区：
文献类型：
--
作者：
KRISHNA, RG;WOLD, F
为了探索底物一级结构的具体特征如何影响兔肌肉酰氨酰基肽水解酶 (EC 3.4.19.1) 的活性，制备了许多在特定位置含有特定氨基酸取代的乙酰化肽，并将其作为水解酶的底物进行比较。主要变体是 D-Ala、Pro 和正电荷（His、Arg、Lys）；此外，还以不太系统的方式研究了肽长度的影响。底物通过肽的直接乙酰化、通过用乙酰氨基酸或乙酰肽延伸N-末端、活化为N-羟基琥珀酰亚胺酯、或者通过从天然存在的乙酰化蛋白质中分离N-末端肽来制备。结果发现，待裂解键两侧（位置 1 和 2）的 D-Ala 完全抑制酶活性，而位置 3 或 4 具有 D-Ala 的乙酰化肽与含有 L-Ala 的肽一样是良好的底物。第 2 位带有 Pro 的肽也没有活性，并且大多数第三位带有 Pro 的肽都是非常差的底物；只有肽 Ac-AAP 具有相当高的活性（Ac-AAA 的 30%），如果 C 末端存在其他残基（Ac-AAPA、Ac-AAPAA），则活性会降低至 1-2%。位置2、3、4、5和6处的正电荷的存在使水解酶活性强烈降低，其随电荷与N末端的距离的变化而变化，从0到15-20%，是用不带正电荷的参考肽获得的速率。 His 在高 pH 下的去质子化产生了优异的底物，通过乙酰化或更好的琥珀酰化去除 Lys 的正电荷也提高了底物质量，证明正电荷是抑制的原因。通常发现长肽（10-29 个残基）是较差的底物，特别是当它们含有正电荷和 Pro 时。更好的长肽底物没有这些残基，而是含有负电荷。对 100 多种乙酰化蛋白质 N 端序列的调查显示，其中约 95% 在前 10 个残基中含有 Pro 和/或带正电荷的氨基酸，表明这些残基可能是体内水解酶作用的天然抑制剂。除了上述残基对底物质量的特定和巨大影响之外，似乎每个肽的整体序列也存在一般影响，并且单个残基的特定影响受到它们表达的环境（上下文）的显着调节。
In an attempt to explore how specific features of the substrate's primary structure may affect the activity of rabbit muscle acylaminoacyl-peptide hydrolase (EC 3.4.19.1), a number of acetylated peptides containing specific amino acid replacements in specific positions were prepared and compared as substrates for the hydrolase. The principal variants were D-Ala, Pro, and positive charges (His, Arg, Lys); in addition, the effect of the length of the peptide was also investigated in a less systematic manner. The substrates were either prepared by direct acetylation of peptides, by extension of the N-terminus with acetylamino acids or acetylpeptides, activated as N-hydroxysuccinimide esters, or by isolation of the N-terminal peptides from naturally occurring acetylated proteins. It was found that D-Ala on either side of the bond to be cleaved (positions 1 and 2) completely inhibited the enzymatic activity, whereas acetylated peptides with D-Ala in positions 3 or 4 were as good substrates as those containing L-Ala. Peptides with Pro in positions 2 were also inactive, and most of the peptides with Pro in the third position were very poor substrates; only the peptide Ac-AAP gave reasonably high activity (30% of Ac-AAA), which was reduced to 1-2% if additional residues were present at the C-terminus (Ac-AAPA, Ac-AAPAA). The presence of a positive charge in positions 2, 3, 4, 5, and 6 gave strong reduction in hydrolase activity varying with the charge's.distance from the N-terminus from 0 to 15-20% of the rates obtained with the reference peptides without positive charges. Deprotonation of His at high pH generated excellent substrates, and removal of the positive charges of Lys by acetylation or, even better, succinylation also gave improved substrate quality, demonstrating that the positive charges are responsible for the inhibition. Long peptides (10-29 residues) were generally found to be poor substrates, especially when they contained positive charges and Pro. The better long peptide substrates do not have these residues, but contain negative charges instead. A survey of the N-terminal sequences of more than 100 acetylated proteins revealed that about 95% of them have Pro and/or positively charged amino acids among the first 10 residues, suggesting that these residues may be natural inhibitors of hydrolase action in vivo. In addition to the specific and large effect of the residues described above on substrate quality, it also appears that there is a general effect of the overall sequence of each peptide, and that the specific effects of individual residues are modulated significantly by the environment (context) in which they are expressed.