Quantum Chemistry Calculation of Thermophilic Protease PH1704 Allosteric Center and Mutant Dynamics†

doi:10.7503/cjcu20130792

Abstract

Abstract:

The PH1704 allosteric sites were studied with the quantum chemistry analysis and the crystal structure analysis. The results show that key residues are Arg113, Tyr120 and Asn129. Tyr120 is connected with nucleophilic residues Cys100 by a hydrogen bond, participates in enzyme nucleophilic catalyst, and is validated by fixed-point mutation of molecular biology experiments. The structures of four building protein of DJ-1 superfamily show that the 120 site locates in the substrate binding pocket in the subunit interface and affects the enzyme activity of the protein. The k_cat/k_m(L·μmol^-1·min^-1) value of mutant R113T/Y120P/N129D is six times higher than that of the wild-type and the Hill coefficient changes from 0.86(wild type) to 1.3 with negative cooperativity disappearing. The main reason is that the residue of 120 site changes from Tyr to Pro, and the hydrogen bonds between Tyr120 and Cys100 are broken, thus its nucleophilic attacking resis-tance decreases, which causes the enzyme activity to increase. The mutations of 113 and 129 sites lead to the detachment of the anionic allosteric agent, thus the negative cooperativity disappears. This work predictes the allosteric site of thermophilic protease by quantum chemistry and crystal structure analysis and provides a solid foundation for further research on the allosteric enzyme of DJ-1 superfamily.

Key words: Thermophilic protease, Quantum chemistry calculation, Allosteric center, Site-mutant

CLC Number:

O641
O623

TrendMD:

ZHAN Dongling, GAO Nan, HAN Weiwei, FENG Yan. Quantum Chemistry Calculation of Thermophilic Protease PH1704 Allosteric Center and Mutant Dynamics^†[J]. Chem. J. Chinese Universities, 2014, 35(1): 146.

Figures/Tables 11

Fig.1 Structures of Lon(A) and PH1704(B)

Fig.2 Crystal structure of PH1704(A) SO42- binding mode in the crystal structure; (B) allosteric site(Arg113, Asn129) and catalytic site(Cys100, His101 and Glu474).

Fig.3 Kinetic curve of wild type PH1704 with substrate AAFR-AMC

Fig.4 Spacial structrue and orbital distribution of L112—A130 fragments(A) Spacial structure of L112—A130; (B) HOMO orbital; (C) LUMO orbital.

Table 1 Molecular orbital distributions

Orbital	HOMO-3	HOMO-2	HOMO-1	HOMO	LUMO	LUMO+1	LUMO+2	LUMO+3
Energy level/eV	-3.8790	-3.6584	-3.6402	-3.1192	-2.9785	-2.8413	-2.6656	-2.5014
Main component	Asp126	Asp126	Asp125	Arg113	Tyr120	Arg115	Arg115	Gly114

Fig.5 Molecular docking of PH1704 with substrate AAFR-AMC(A) AAFR-AMC in the active pocket of PH1704; (B) important residues in the binding of AAFR-AMC and PH1704.

Fig.6 Cα RMSD plot of complex PH1704-AAFR-AMCa. Mutant; b. wide type(WT).

Fig.7 Sequence alignment of PH1704 with other members in the DJ-1 superfamilyPfpI,Pyrococcus furiosus protease I(90.4%, percentage identity); NA2, an intracellular protease from Pyrococcus sp. NA2(88%); GE5, an intracellular protease from Pyrococcus abyssi GE5(86%); EJ3, an intracellular protease from Thermococcus gammatolerans EJ3(85.0%); 3L18, an intracellular protease from Thermococcus onnurineus NA1(78%); YP372, an intracellular protease from Methanohalobium evestigatum Z-7303(61%). According to the decending order, the homology levels are highlighted by dark blue, pink and sky blue, respectively.

Table 2 Assessment of protein structure

Protein	Procheck(Percentage identity, %)				Errat score
PfpI	Core(90.2)	Allow(9.1)	Gener(0.0)	Disall(0.8)	97.046
NA2	Core(89.4)	Allow(9.2)	Gener(0.7)	Disall(0.7)	92.194
GE5	Core(88.0)	Allow(10.6)	Gener(0.7)	Disall(0.7)	94.515
3L18	Core(88.8)	Allow(9.8)	Gener(0.7)	Disall(0.7)	98.301

Fig.8 Molecular docking of four proteins with substrate AAFR-AMC(A) 3L18; (B) PfpI; (C) NA2; (D) GE5.

Table 3 Kinetic parameters for hydrolysis substrates of AAFR-AMC

Enzyme	k_cat/min^-1	k_m/(μmol·L^-1)	h	(k_cat/k_m)/(L·μmol^-1·min^-1)
WT	0.11	10	0.86	0.01
R113T/Y120P/N129D	0.79	13.05	1.3	0.06

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