Kumar Ajit, Rao Mala
Division of Biochemical Sciences, National Chemical Laboratory, Pune- 411-008, India.
Biochim Biophys Acta. 2010 May;1800(5):526-36. doi: 10.1016/j.bbagen.2010.01.014. Epub 2010 Feb 6.
Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. The majority of chitinase inhibitors reported are natural products like argifin, argifin linear fragments, argadin, allosamidin and disulfide-cyclized peptides. Here, we report a novel peptidic inhibitor API (Aspartic Protease Inhibitor), isolated from Bacillus licheniformis that inhibits chitinase A (ChiA) from Serratia marcescens.
The binding affinity of API with ChiA and type of inhibition was determined by the inhibition kinetics assays. Fluorescence and CD spectroscopic analysis and chemical modification of API with different affinity reagents elucidated the mechanism of binding of API with ChiA.
The peptide has an amino acid sequence N-Ile(1)-Cys(2)-Glu(3)-Ala(4)-Glu(5)-His(6)-Lys(7)-Trp(8)-Gly(9)-Asp(10)-Tyr(11)-Leu(12)-Asp(13)-C. The ChiA-API kinetic interactions reveal noncompetitive, irreversible and tight binding nature of API with I(50) = 600 nM and K(i)= 510 nM in the presence of chromogenic substrate p-nitrophenyl-N,N'-diacetyl-beta-chitobioside[p-NP-(GlcNAc)(2)]. The inhibition progress curves show a two-step slow tight binding inhibition mechanism with the rate constant k(5) = 8.7 +/- 1 x 10(-3) s(-1) and k(6) = 7.3 +/- 0.6 x 10(-5) s(-1). CD-spectra and tryptophanyl fluorescence analysis of ChiA incubated with increasing API concentrations confirms conformational changes in enzyme structure which may be due to irreversible denaturation of enzyme upon binding of API. Chemical modifications by WRK abolished the anti-chitinase activity of API and revealed the involvement of carboxyl groups in the enzyme inactivation. Abolished isoindole fluorescence of OPTA-labeled ChiA demonstrates the irreversible denaturation of ChiA upon incubation with API for prolonged time and distortion of active site of the enzyme.
The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.
几丁质酶抑制剂具有作为杀真菌剂、杀虫剂和抗哮喘药的化疗潜力。报道的大多数几丁质酶抑制剂是天然产物，如阿吉芬、阿吉芬线性片段、阿加丁、别洛沙米丁和二硫键环化肽。在此，我们报道了一种从地衣芽孢杆菌中分离出的新型肽类抑制剂API（天冬氨酸蛋白酶抑制剂），它能抑制粘质沙雷氏菌的几丁质酶A（ChiA）。
通过抑制动力学测定确定API与ChiA的结合亲和力及抑制类型。利用荧光和圆二色光谱分析以及用不同亲和试剂对API进行化学修饰，阐明了API与ChiA的结合机制。
该肽的氨基酸序列为N - Ile(1)-Cys(2)-Glu(3)-Ala(4)-Glu(5)-His(6)-Lys(7)-Trp(8)-Gly(9)-Asp(10)-Tyr(11)-Leu(12)-Asp(13)-C。在生色底物对硝基苯基 - N,N'-二乙酰 - β - 壳二糖[p - NP-(GlcNAc)(2)]存在的情况下，ChiA - API的动力学相互作用显示API具有非竞争性、不可逆和紧密结合的性质，I(50) = 600 nM，K(i)= 510 nM。抑制进程曲线显示出两步缓慢紧密结合抑制机制，速率常数k(5) = 8.7 +/- 1 x 10(-3) s(-1)，k(6) = 7.3 +/- 0.6 x 10(-5) s(-1)。对与不断增加浓度的API一起孵育的ChiA进行圆二色光谱和色氨酸荧光分析，证实了酶结构的构象变化，这可能是由于API结合后酶的不可逆变性所致。用WRK进行化学修饰消除了API的抗几丁质酶活性，并揭示了羧基在酶失活中的作用。OPTA标记的ChiA的异吲哚荧光消失表明，与API长时间孵育后ChiA发生不可逆变性，且酶的活性位点发生扭曲。
这些数据提供了有用信息，可能有助于开发类药物的、基于天然产物的几丁质酶抑制剂。
