Utilization of synthetic peptides to evaluate the importance of substrate interaction at the proteolytic site of Escherichia coli Lon protease.
Utilization of synthetic peptides to evaluate the importance of substrate interaction at the proteolytic site of Escherichia coli Lon protease.
复制标题
利用合成肽来评估在大肠杆菌蛋白酶的蛋白水解位点底物相互作用的重要性。
DOI：
10.1016/j.bbapap.2009.02.015
复制
发表时间：
2009-09
期刊：
影响因子：
--
通讯作者：
Lee I
中科院分区：
文献类型：
--
作者：
Patterson-Ward J;Tedesco J;Hudak J;Fishovitz J;Becker J;Frase H;McNamara K;Lee I
Lon，也称为蛋白酶La，是一种ATP依赖性蛋白酶，其功能是降解许多非结构化蛋白质。目前，对Lon蛋白水解位点的底物决定簇知之甚少。利用构成内源性蛋白底物λN不同区域的合成肽，我们证明了大肠杆菌Lon的蛋白水解位点表现出一定程度的局部序列特异性。使用丙氨酸位置扫描的方法，我们发现了一组不连续的基板周围的易切割的Lon切割位点的模型肽基板，其功能是影响Lon的肽酶活性的kcat的决定因素。我们进一步研究了蛋白水解失活的Lon突变体S679A在ADP或AMPPNP的存在和不存在下，通过二维核磁共振光谱的肽相互作用的模式，并发现，蛋白质和肽之间的结合相互作用的变化与核苷酸结合的酶。这一观察结果提示了底物易位步骤，这可能限制了蛋白水解反应的周转。还检查了所鉴定的底物决定簇对Lon对λN和截短λN突变体的ATP依赖性降解动力学的贡献。我们的研究结果表明，Lon可能在λN中识别出许多不连续的底物决定簇，从而实现底物混杂。
Lon, also known as protease La, is an ATP-dependent protease functioning to degrade many unstructured proteins. Currently, very little is known about the substrate determinants of Lon at the proteolytic site. Using synthetic peptides constituting different regions of the endogenous protein substrate λN, we demonstrated that the proteolytic site of Escherichia coli Lon exhibits a certain level of localized sequence specificity. Using an alanine positional scanning approach, we discovered a set of discontinuous substrate determinants surrounding the scissile Lon cleavage site in a model peptide substrate, which function to influence the kcat of the peptidase activity of Lon. We further investigated the mode of peptide interaction with the proteolytically inactive Lon mutant S679A in the absence and presence of ADP or AMPPNP by 2-dimensional nuclear magnetic resonance spectroscopy, and discovered that the binding interaction between protein and peptide varies with the nucleotide bound to the enzyme. This observation is suggestive of a substrate translocation step, which likely limits the turnover of the proteolytic reaction. The contribution of the identified substrate determinants towards the kinetics of ATP-dependent degradation of λN and truncated λN mutants by Lon was also examined. Our results indicated that Lon likely recognizes numerous discontinuous substrate determinants throughout λN such to achieve substrate promiscuity.
登录
查看更多内容
影响因子：
10.5
作者：
Gur, Eyal;Sauer, Robert T.
通讯作者：
Sauer, Robert T.
影响因子：
15
作者：
Farbman, Mary E.;Gershenson, Anne;Licht, Stuart
通讯作者：
Licht, Stuart
影响因子：
3.2
作者：
GOTTESMAN, S;ZIPSER, D
通讯作者：
ZIPSER, D
DOI：
10.1073/pnas.80.2.358
发表时间：
1983-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
MIZUSAWA, S;GOTTESMAN, S
通讯作者：
GOTTESMAN, S
DOI：
10.1073/pnas.78.8.4931
发表时间：
1981-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
CHUNG, CH;GOLDBERG, AL
通讯作者：
GOLDBERG, AL