분자모델링을 이용한 유기용매에서의 효소 안정성 연구

연영주, 유영제

서울대학교 공과대학 응용화학부

Molecular Modeling of Enzyme Stability

in Organic Media

Young Joo Yeon, Young Je Yoo

School of Chemical Engineering, Seoul National University

Introduction

The use of enzymes in organic solvents offers possibilities for new industrial applications. However, the direct contact of organic solvent with enzyme can result in conformational change, which finally lead to deactivation and destabilization [1]. For this reason, there have been numerous researches for the enhancement of enzyme activity and/or stability in organic solvents. Using the methods of directed evolution and site-directed mutagenesis, several successful results were reported [5-8]. However, these methods are kinds of trial-and-error. Therefore, a predictive method based on theoretical study is required.

In present study, the difference between structure of the organo-philic enzymes (which are active and/or stable in the organic solvents) and the counterpart enzymes (the common enzyme) were analyzed using molecular simulation. The solvation free energy was employed as a key parameter for the comparative analysis and for the development of a new predictive simulation method.

Materials and Methods

The structure of the model enzymes were basically obtained from the PDB web site (http://www.rcsb.org) - subtilisin DY (1MEE), □-lytic protease (2ALP), PST-01 protease (1EZM), Thermolysin (1LNF). However, for the structures that were not found in PDB web site, amino acid substitution was performed using the BIOPOLYMER module of SYBYL package (Tripos Inc.).

After the substitution, energy minimization was performed using Minimize command of SYBYL program for the refinement of the structures. Ramachandran plot [2] analysis was performed by PROCHECK program [3] for the validation of minimized structure.

Solvation free energy [4] of each enzyme set was calculated using ProTable of SYBYL package, and the results between organo-philic enzymes and the counterpart enzymes were compared.

Results and Discussion

The Ramachandran plots that were generated by PROCHECK showed that all the minimized structures were acceptable for molecular calculation - no residue in the "disallowed" region was found.

In order to calculate solvent exposed hydrophobicity of enzyme, solvation free energy was calculated. Solvation free energy is defined as follows [4].

[eqn: rm DELTA G_s {}={} DELTA delta (C) SUM from { C {}atoms{} i} (A_i - A^r_i ){} +{} DELTA delta (N/O) SUM from { N,O {}atoms{} i} (A_i - A^r_i )]

[eqn: rm+{}DELTA delta (O^- ) SUM from { O^- {}atoms{} i} (A_i - A^r_i ){}+{} DELTA delta (N^+ ) SUM from { N^+ {}atoms{} i} (A_i - A^r_i )]

[eqn: rm+{}DELTA delta (S) SUM from { S {}atoms{} i} (A_i - A^r_i )]

□Gs : solvation free energy

i : expression for the ith atom

A : solvent accessible solvent area

Ar : A in reference state

□□: atomic solvation parameter

□-Lytic protease and Subtilisin E were selected as counterpart enzymes and their SDM (site-directed mutagenesis) mutants were selected as organo-philic enzymes. The SDM mutants were obtained from the substitution of charged surface residue with more hydrophobic ones [5, 6]. From the calculation of solvation free energy, it was found that all the organo-philic enzymes (except for the Asn218 → Ser mutant) have higher values of solvation free energy than the counterpart enzymes (Table 1,2).

Table 1. Solvation free energy of □-lytic protease and the SDM mutants (obtained from surface charge substitution)
Type	Variant	Solvation Free Energy (kcal/mol)	t1/2 (min) in 84% DMF, 30℃[5]
Counterpart	□-Lytic protease (wt)	-2261.23	23
Organo-philic	Arg65 → Glu	7101.27	138
Organo-philic	Arg65 → Ser	7376.98	32
Organo-philic	Arg65 → Leu	7498.27	34
Organo-philic	Arg65 → Ile	6801.50	31
Organo-philic	Arg120A → Leu	10040.48	96
Organo-philic	Arg120A → Tyr	9301.20	71
Organo-philic	Arg120A → Phe	10414.12	59
Organo-philic	Arg65 → Glu + Arg120A →Leu	11791.15	626

Table 2. Solvation free energy of subtilisin E and the SDM mutants (obtained from surface charge substitution)
Type	Variant	Solvation Free Energy (kcal/mol)	t1/2 (h) in 80% DMF, 30℃[6]
Counterpart	Subtilisin E (wt)	17977.94	5.7
Organo-philic	Asp248 → Asn	18047.05	10.2
Organo-philic	Asp248 → Ala	18888.60	10.3
Organo-philic	Asp248 → Leu	20048.66	11.1
Organo-philic	Asn218 → Ser	17374.98	10.2
Organo-philic	Asp248 → Asn + Asn218 →Ser	18367.81	19.2

The solvation free energies of organo-philic enzymes obtained from directed evolution (Table 3), the SDM mutants obtained from the formation of disulfide bonds (Table 4) and the naturally organo-philic enzyme (Table 5) were also calculated and compared with the that of counterparts, and the same results were obtained. In Table 5, the counterpart enzyme of Cys58 → Gly was compared as the mutant obtained from the deletion of Cys30-Cys58 disulfide bond.

From these results, it was found that almost all the organo-philic enzymes tested have higher solvation free energy than the counterpart enzymes. This shows the possibility that the solvation free energy could be used as a key parameter for the development of a new predictive simulation method for the stabilization of enzyme in organic solvents.

* PC3 : 10 mutants of subtilisin E    * 13M : 12 mutants of subtilisin E

Table 3. Solvation free energy of subtilisin E(wild type), PC3 and 13M (obtained from directed evolution)
Type	Variant	Solvation Free Energy (kcal/mol)	kcat/Km (M-1s-1×103) in 60% DMF, 37℃[7]
Counterpart	Subtilisin E	17977.94	0.0014
Organo-philic	PC3	19886.51	2.2
Organo-philic	13M	20127.37	6.6]

* The concentration of DMF and ethanol that inhibits  50% subtilisin          activity(IC50) was determined at 37℃.

* CSS : 2 mutants of subtilisin E

* DSS : 4 mutants of subtilisin E

* LDSS : 5 mutants of subtilisin E

Table 4. Solvation free energy of subtilisin E and the SDM mutants (obtained from disulfide bonds formation)
Type	Variant	Solvation Free Energy (kcal/mol)	IC50(%)[8]
			DMF	Ethanol
Counterpart	Subtilisin E (wt)	17977.94	9.0	7.5
Organo-philic	CSS	18474.92	13.1	11.6
Organo-philic	DSS	18406.87	14.7	12.3
Organo-philic	LDSS	18140.24	16.4	11.6

Table 5. Solvation free energy of PST-01 protease (naturlly organo-philic) and the counterpart enzymes
Type	Variant	Solvation Free Energy (kcal/mol)	t1/2 (d) in 33% DMF, 30℃
Counterpart	Thermolysin	-6447.03	0.9[1]
Counterpart	Cys58 → Gly	-3343.24	3.5[9]
Organo-philic	PST-01 protease	947.99	>100[9]

References

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[5] P. Martinez, F. H. Arnold, J. Am. Chem. Soc. 113, 6336 (1991).

[6] P. Martinez, M. E. V. Dam, A. C. Robinson, K. Chen, F. H. Arnold,           Biotechnol. Bioeng. 39, 141 (1992).

[7] L. You and F. H. Arnold, Protein Engineering 9, 77 (1994).

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