The effect of organic compounds on the chitinase activity the dissociation constant calculation of the "Chitinase-metallic ion" complex

Journal of Chemistry, Vol. 41, No. 2, P. 110 - 114, 2003<br /> <br /> <br /> The effect of organic compounds on the chitinase<br /> activity The dissociation constant calculation of<br /> the "Chitinase-metallic ion" complex<br /> Received 12-3-2002<br /> Naoyoki Taniguchi , Yoshitaka Ikeda1, Tran Dinh Toai2<br /> 1<br /> <br /> 1<br /> Department of Biochemistry, Osaka University Medical School, Japan<br /> 2<br /> Institute of Chemistry, National Center for Natural Science and Technology of Vietnam<br /> <br /> <br /> Summary<br /> The effect of some organic compounds on the activity of chitinase from Serratia marcescens<br /> has been investigated. Among these compounds, 1,10-phenanthroline was found to be an<br /> inhibitor and ethanol - an activator of the chitinase activity; - ’ dipyridyl and Zn2+ do not take<br /> any effect on the chitinase activity.<br /> In this investigation, the dissociation constant of an chitinase-metalic ion complex (K1) and<br /> the inhibition constant (K2) have been estimated with K1 = 0.1 mcM and K2 = 2.667 mM.<br /> <br /> <br /> I - Introduction mechanism is important for the design of<br /> understanding properties of this enzyme [3, 4].<br /> Chitinases (EC 3.2.1.4) belong to numbers A typical colorimetric substrate for<br /> of glycosyl hydrolases. These numbers related assaying exochitinase activity is p-nitrophenyl -<br /> enzymes (482 sequences) corresponding to 52 N-acetyl-beta-D-glucosaminide (pNP-beta-<br /> EC entries of the I.U.B classification system GlucNAc) and for assaying endochitinase<br /> have been classified into 45 families. Chitinases activity is p-nitrophenyl- -D-N,N',N'-triacetyl-<br /> catalyze the hydrolysis of chitin, an insoluble chitotrise, respectively [5].<br /> linear (1,4)-linked polymer of N-acetyl-<br /> The chitinase activity is effected by many<br /> glucosamine (GlcNAc), and are critical for the<br /> organic compounds (argifin, allosamidin,<br /> normal development of insects.<br /> argadin [6, 7]), heavy metal ions (Ag+, Hg2+,<br /> On the basis of amino acid sequence, five Fe2+, Fe3+), and chemical modified agents (NaI,<br /> known classes of Chitinases are grouped into NBS).<br /> two glycosyl hydrolase families [1, 2].<br /> The exochitinase is a metalloenzyme, and<br /> Family 18 consists of class III and class V its activity is inhibited by 1,10-phenanthroline.<br /> Chitinases found in a wide range of organisms The zinc plays an important role in functioning<br /> including bacteria, plants, animals, fungi and of exochitinase [8].<br /> viruses. Family 19 consists of class I, II and IV<br /> In order to understand the mechanism of<br /> Chitinases and is found only in plants.<br /> chitinase action, we had tried to find a<br /> The enzyme structures and hydrolysis dissociation constant of a "chitinase-metallic<br /> mechanisms for two families appear to be quite ion" complex and an influence of some organic<br /> different. The knowledge of the reaction compounds on the chitinase activity.<br /> <br /> 110<br /> II - Determination of dissociation equation (1 - 4), if these constants K1, K2 are<br /> constant of a "enzyme-metallic known. There are several solutions to determine<br /> ion" complex the dissociation constant of "enzyme-metallic<br /> ion" complex<br /> 1. Enzymatic kinetic method for Determin- A total initial concentration of enzyme can<br /> ation of dissociation constant of an be summarized:<br /> "enzyme-metallic ion" complex [E]0 = [E] + [E*] (5)<br /> It is suggested that, the enzyme will lose its Equation (2 - 5) can be summarized by the<br /> activity if metallic ion goes out of the enzyme following:<br /> active center. It is represented by the following<br /> scheme:<br /> K1 [E ] = [E ] ([E ] [E ])K/(1 + [I ] / K<br /> * 1<br /> <br /> )<br /> (6)<br /> 0 2<br /> E = E* + M (1)<br /> There: From (5) [E*] = [E0] – [E] are known, so:<br /> E is an active form of the enzyme,<br /> K1<br /> E* is a non-active form of the enzyme or [E 0 ] [E ] = [E ] (7)<br /> ([E 0 ] [E ]) / (1 + [I ] / K 2 )<br /> M is a metallic ion (zinc)<br /> K1 is dissociation constant of a "enzyme- Dividing both influences of the equation (7)<br /> metallic ion" complex. on [E0], we obtain the following equation:<br /> A dissociation constant can be represented ([E0 ] [E ])2 K1 [I ]<br /> by the following equation: = × 1+ (8)<br /> [E0 ][E ] [E0 ] K2<br /> K1 =<br /> [E ]× [M ]<br /> *<br /> (2) Squaring the left of equation (7)<br /> [E ] [E0 ]2 + [E ]2 2[E0 ][E ] = [E ]K1 (1 + [I ] / K 2 )<br /> Because zinc plays an important role in<br /> or<br /> functioning of exochitinase [13], it may take<br /> part in enzyme active center. Therefore, E* can [E ]2 [E ]{K 1 (1 + [I ] / K 2 ) + 2[E 0 ]} + [E 0 ]2 = 0<br /> be activated by this metallic ion (zinc) and the (9)<br /> inhibition will occur by using the compound in Let [E] = X ; {K 1 (1 + [I ] / K 2 ) + 2[E 0 ]} = b ;<br /> combination with ion to produce complex. This<br /> process can be represented by the following<br /> [E0 ]2 = C<br /> scheme: We have an equation of second degree:<br /> K2 aX2 + bX + C = 0, in which a is 1.<br /> M + I = MI (3)<br /> There: It is enabling to solve this equation to find<br /> out a solution:<br /> I is an organic compound as an inhibitor<br /> M I is an "organic compound-metallic ion" [E ] = X 1 ; X 2 = 2a ± b 2 + 4ac<br /> complex and then compare the experimentation to get<br /> K2 is the inhibition constant, can be meaningful solutions.<br /> represented by the following equation: The solutions indicate the value of enzyme<br /> <br /> K2 =<br /> [M ] × [I ] (4)<br /> strength depends on the kinetic parameters in<br /> reaction processes.<br /> [MI ] In order to reduce the complexity, it is<br /> An active enzymatic conformation [E] in suggested: [E] is much smaller than [E0] in the<br /> so [E ] > 2[E 0 ] We have used an artificial substrate, p-<br /> nitro-phenyl-N-acetylglucosamine, for<br /> From equation (11) we obtain: enzyme’s reaction and to measure the enzyme’s<br /> [E 0 ]2 activity and kinetic parameters. A unit of<br /> [E ] = (12)<br /> K 1 (1 + [I ] / K 2 ) chitinase is understood as an amount of<br /> enzyme, which catalyzed the release of 1<br /> In the other shape: micromole of soluble p-nitro-phenol from p-<br /> [E0 ] K1 [I ] nitro-phenyl-N-acetylglucosamine in 1 min. at<br /> = 1+ (13) 30oC.<br /> [E ] [E0 ] K2<br /> The product of enzyme’s reaction was<br /> It is obvious that, a speed of enzyme measured by its absorption at 420 nm wave<br /> reaction is proportionate to an enzyme with extinction coefficient 18,000 m-1cm-1.<br /> concentration with some coefficient : V =<br /> [E]. Accordingly, equation (8) is substituted III- RESULTS AND DISCUSSION<br /> by the following one:<br /> 1. The inhibition of chitinase by the organic<br /> (V0 Vi )<br /> 2<br /> K<br /> = 1 × 1+<br /> [I ] (14)<br /> compounds 1,10-phenanthroline<br /> V0Vi [E0 ] K2 The inhibition of chitinase from Serratia<br /> marcescens was investigated using the 1,10-<br /> An equation (13) is substituted by the phenanthroline as an inhibitor. The 1,10-<br /> following one: phenanthroline inhibits strongly the chitinase<br /> V0 K<br /> = 1 1+<br /> [I ] (15)<br /> activity (table 1).<br /> Vi [E 0 ] K2 Based on this investigation (by creating a<br /> graph V0/Vi = f([I]) (Fig. 1) the dissociation<br /> It is possible to determined K1 and K2 by constant of an enzyme-metallic ion complex<br /> creating a graph (V0-Vi)2/V0Vi = f([I]) (14) or (K1) and the inhibition constant (K2) have been<br /> V0/Vi = f([I]) (15), when the total initial estimated.<br /> concentration of enzyme [E0] was known. K1 = 0.1 mcM, and K2 = 2.667 mM.<br /> <br /> Table 1: The inhibition effect of 1,10-phenanthroline [I] on the activity of chitinase<br /> from Serratia marcescens. [E0] = 1 x 10-6 M<br /> <br /> No 1 2 3 4 5 6<br /> [I], µM 10 5 2 1 0.5 0<br /> 6<br /> V.10 , M/min 1.8 4.8 8.9 7.6 6.0 9.6<br /> V0/Vi 5.3 2.0 1.6 1.3 1.1 1.0<br /> <br /> 112<br />  Figure 1: Dissociation constant determination of a chitinase-metallic ion complex (K1)<br /> 2. The effect of on the chitinase activity<br /> The effect of the other compounds on the activity of chitinase from Serratia marcescens<br /> has been found. Ethanol has been found to be one of these compounds, such as activator and brings<br /> into effect on the chitinase activity (table 2); - ’ dipyridyl and Zn2+ do not take any effect.<br /> <br /> Table 2: The effect of ethanol on the chitinase activity<br /> No 8 7 6 5 4 3 2 1<br /> [A], mM 1000 360 120 60 30 15 7.50 0<br /> D, 420 nm 0.53 0.31 0.51 0.46 0.35 0.33 0.42 0.31<br /> 6<br /> V.10 , M/min 10.9 5.70 9.30 9.20 6.50 6.10 7.80 5.50<br /> VA/V0 1.69 1.00 1.62 1.47 1.13 1.05 1.35 1.00<br /> <br /> Acknowledgements: school, Osaka University, Japan for taking<br /> We are owned to JSPJ for providing the care and all the best conditions for completing<br /> best conditions for our working-time in Japan. the work.<br /> We are especially grateful to Prof. Dr. This paper was financial supported by<br /> Naoyoki Taniguchi, who contribute us many capital research program.<br /> good scientific idea, and provide us with the<br /> best conditions for successful completing the References<br /> work.<br /> 1. 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