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CD Spectra. CD spectroscopy was used to study porcine lipase conformational changes

 before and after EGCG binding.The CD spectra of lipase exhibited a negative band in the

ultraviolet region at 20 nm !"igure #$% which is characteristic of &'helical structures in

 protein.() *fter EGCG binding% EGCG+lipase complexes had greater absolute , values than

did native lipase in the ultraviolet region at 20 nm% which suggests the presence of some &'

helical structures in the protein. -e used E/C1( in DC34-E5 to determine thesecondary structure. n complexation of lipase with EGCG% the &'helical content of lipase

changed from 26 to 7#8% with a

decrease in 9'sheets from 27 to 678% and the unordered structure content changed from ( to

2#8 !Table 2$. Thus% the binding of EGCG on lipase caused conformational changes in the

en:yme.

CD spe;tros;opi diguna;an untu; mempela<ari perubahan ;onformasi lipase babi sebelum

dan sesudah i;atan EGCG .pe;trum CD lipase menun<u;;an pita negatif di daerah

ultraviolet pada pan<ang gelombang 20 nm !Gambar #$% yang merupa;an ;ara;teristi; dari

stru;tur &'heli;s pada protein. etelah EGCG mengi;at% ;omple;s EGCG'lipase memili;i

nilai , absolut daripada lipase asli di daerah ultraviolet pada 20 nm% yang menun<u;;an;eberadaan beberapa stru;tur &'heli;s dalam protein. =ami mengguna;an E/C1( di

DC34-E5 untu; menentu;an stru;tur se;under. >ada ;omple;sasi lipase dengan EGCG%

;andungan &'heli;s dari lipase berubah dari 26 ;e 7#8% dengan penurunan 9'sheet dari 27 ;e

678% dan ;onten stru;tur yang tida; tersedia berubah dari ( ;e 2#8 !Tabel 2$. Dengan

demi;ian% pengi;atan EGCG pada lipase menyebab;an perubahan ;onformasi dalam en:im.

Docking Studies. ?olecular doc;ing studies were used to substantiate our experimental

findings. The doc;ing results showed that EGCG was surrounded by @al26% Gln22% le20%

*rg6A6% Gln6% Gln220% Cly6B% >ro6)% Thr6#% and Gln220 in lipase !"igure )$.

3ydrogen'bonding interaction appears plausible between @al26% Gln6% and Gln220. These

extensive hydrogen'bonding interactions might play an important role in

increased binding affinity of EGCG and lipase. The calculated G for EGCG+lipase binding

was +27.)) ; mol+6% which was close to the TC'measured values of G. The catalytic sites

of porcine lipase are residues er6A7% 3is7(B% and *sp(20.( "igure ) shows that these

residues do not surround EGCG. *s well% ;inetics findings for lipase demonstrated that

EGCG inhibition of lipase was noncompetitive. Therefore% EGCG inhibited porcine lipase

activities by not binding the catalytic sites of lipase. The conformation of an en:yme depends

on noncovalent and covalent interactions among the en:yme amino acid residues. The

activity of an en:yme will be changed if its specific conformation changes. The main

mechanism of tea polyphenol binding proteins is considered noncovalent interaction.2(%2A

-e found that the interaction of EGCG and lipase was noncovalent. 1oncovalent binding isoften wea; and nonspecific% but a combination of many noncovalent bonds may alter the

conformation and function of the protein.(A The internal noncovalent interactions of the

 peptide chain may be altered or even destroyed when a certain compound is added to a

 protein solution.(A nevitably% the function of the protein is affected when it binds with any

chemical substance. The conformational mobility of the protein structure influences the

catalytic activity of en:ymes.70

Studi docking. tudi doc;ing mole;uler diguna;an untu; mendu;ung temuan e;sperimental

;ami. 3asil doc;ing menun<u;;an bahwa EGCG di;elilingi oleh @al26% Gln22% le20%*rg6A6% Gln6% Gln220% Cly6B% >ro6)% Thr6#% dan Gln220 di lipase !Gambar )$.

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ntera;si i;atan hidrogen muncul diantara @al26% Gln6% dan Gln220. ntera;si i;atan

hidrogen yang luas mung;in memain;an peran penting dalam pening;atan afinitas

 pengi;atan EGCG dan lipase. G yang dihitung untu; i;atan EGCG'lipase adalah '27%)) ;

mol'6% yang de;at dengan nilai'nilai pengu;uran TC dari G. itus ;ataliti; lipase babi

merupa;an residu er6A7% 3is7(B% dan *sp(20.( Gambar ) menun<u;;an bahwa residu ini

tida; mengelilingi EGCG.erta%penemuan ;ineti;a untu; lipase menun<u;;an bahwa penghambatan EGCG dari lipase adalah non;ompetitif. leh ;arena itu% EGCG menghambat

a;tivitas lipase babi dengan tida; mengi;at situs ;ataliti; lipase. =onformasi en:im

tergantung pada intera;si non ;ovalen dan ;ovalen antara residu asam amino en:im. *;tivitas

en:im a;an berubah <i;a ter<adi perubahan ;onformasi spesifi;. ?e;anisme utama protein

 pengi;at polifenol teh dianggap intera;si non ;ovalen. =ami menemu;an bahwa intera;si

EGCG dan lipase adalah non ;ovalen i;atan non ;ovalen sering;ali lemah dan tida; spesifi;%

tetapi ;ombinasi dari beberapa i;atan non;ovalen dapat mengubah ;onformasi dan fungsi

 protein.ntera;si non ;ovalen internal dari rantai peptida dapat berubah atau bah;an hancur

;eti;a senyawa tertentu ditambah;an ;e larutan protein.Ta; heran% fungsi protein dipengaruhi

;eti;a protein teri;at dengan :at ;imia.?obilitas ;onformasi stru;tur protein mempengaruhi

a;tivitas ;ataliti; en:im.