Abstract
Aspartate kinase (AK) is the key enzyme in the biosynthesis of aspartate-derived amino acids. Recombinant AK was efficiently purified and systematically characterized through analysis under optimal conditions combined with steady-state kinetics study. Homogeneous AK was predicted as a decamer with a molecular weight of ~48 kDa and a half-life of 4.5 h. The enzymatic activity was enhanced by ethanol and Ni2+. Moreover, steady-state kinetic study confirmed that AK is an allosteric enzyme, and its activity was inhibited by allosteric inhibitors, such as Lys, Met, and Thr. Theoretical results indicated the binding mode of AK and showed that Arg169 is an important residue in substrate binding, catalytic domain, and inhibitor binding. The values of the kinetic parameter Vmax of R169 mutants, namely, R169Y, R169P, R169D, and R169H AK, with l-aspartate as the substrate, were 4.71-, 2.25-, 2.57-, and 2.13-fold higher, respectively, than that of the wild-type AK. Furthermore, experimental and theoretical data showed that Arg169 formed a hydrogen bond with Glu92, which functions as the entrance gate. This study provides a basis to develop new enzymes and elucidate the corresponding amino acid production.
Highlights
Aspartate kinase (AK) is the first identified important enzyme in the biosynthesis of several amino acids, such as Lys, Thr, and Met, which are essential to mammals
TTinhhiitsshessttirrnuuacccttuutirrvaaell fddoifirffmfeer,rebennucectenmomasyaaylrterbseursiludt lgitneinwthatehsfefluofculutnucdatutiaontnitohinne aitnhcteitvhee fcoartcmaaltya(Ftlyiicgtieucfrefeifcf3iicecin)e.cnTychyoiofsfAsAtKrKu.c. tural difference may result in the fluctuation in the catalytic efficiency of AK
Recombinant AK from C. pekinense is a member of the AK superfamily
Summary
Aspartate kinase (AK) is the first identified important enzyme in the biosynthesis of several amino acids, such as Lys, Thr, and Met, which are essential to mammals. AK is classified based on subunit organization as a homo-oligomer or heterotetramer. Each αβ dimer contains two lysine binding sites [12], in which one site is exclusively found in the dimer with A and B chains [13,14,15] located at the interface between α and β subunits The presence of this exclusive site indicates that the lysine-binding site in the regulatory region of CgAK performs a vital function in AK allosteric inhibition [16,17]. Int.IJn. Mthol.isScsi.t2u0d15y,,1w6, peadgee–spcargiebe CpAK, a class II AK with allosteric effectors, namely Lys and Thr, and dwfdwfaooeeerbertdwftmoemaerererresretIsmmsipmenrpsaraeoIsiomtifpnnhrnhohratoeityeitryenheshrddtdtttedyihshehrsrdttadeoeaeouhsrngntgraedodrueeeneigeyirdngmngme,esiyuunigmwbpbgp,luloaobwopneaolntrontaoroedtoodnttdraroaefdrtdynwaysrnwaeytncwtfsnifriutrctufrtiiehraunrhbtenishelnsbecGlicGsiotdceGtdiCiltdislouuCioulutpouunee9npe9nAe9,sr2,s2A,s2in,Knoc,no,Koawwfaw,acffm,mtaomhthathhhnheicieieiccclicitslllyshlsrahhyyaeoseesAfffnAAslnnusuuzrszIzrnIrngyIyIgygycc1smA1Am1tmtt6i66ieKoKe9eo9e9mn....n..wwBsTBTBsTfyiahyaihoyahttshisrichsisccstsotaoatohrahmrhlrsmemeleleepeslobsoesibbaseidieinsdtninrdinenuttntiuetrurtaneiriarrinenteagcianiegcsgnineicessbcefieecxenfieifixepexngofnvpcegpeapgvovtcreateoaorloietrvtormtroleliievoesdvmrm,edfeosoeunned,AfedficatnnnnAatAKimitiatnlneoaK.aKmaenlnTle.eln.zayen.hanTdTynlnizLzyhshmdytdyyihisLmsesmsttetyuhshoscaesetdeaternuucoetayocdadatdrnarilpetTyectyydatarathsilopilplTciyrcyvsrad,rashsoiolaaialdirsvnnvs,dtdeaiddaaiaasdad,nntnteeaadddss,, a baasbiassfisorfotrhtehdeedseigsingnofoaf nanalallolostseterircicccoonnttrroollssyysstteemm for aspaarrttaatteebbiioopprroodduucctitoionn
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
