Evidence that Serine Residue is Essential for Enzyme Activity and Identification of $(^3H)$ Diisopropylfluorophosphate labeled Peptide in EcoRI Endonuclease

권세창,조영동,Kwon, Se-Chang,Cho, Young-Dong 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.2

제한 효소 EcoRI은 serine 잔기에 특이적으로 결합하는 diisopropylfluorophosphate (DFP)에 의해 비활성화 되었다. 이러한 비활성화 속도는 $Mg^{++}$과 기질인 DNA에 의해서 감소하였다. DFP의 작용은 제한 효소 EcoRI의 DNA 절단 활성에 영향을 주는 반면에 DNA 결합 활성에는 영향을 주지 않는 것으로 나타났다. 이러한 결과들로부터 제한 효소 EcoRI에서 serine 잔기가 절단 활성에 관여하고 있을 것으로 사료된다. 효소 활성에 관여하는 serine 잔기의 위치를 확인하기 위하여 $[_3H]$-DFP를 사용한 결과 제한 효소 EcoRI의 146-169 사이에 존재하는 serine 잔기임을 알 수 있었다. EcoRI endonuclease is inactivated by diisopropylfluorophosphate (DFP), a serine residue modification reagent. The rate of inactivation is reduced by the substrate DNA and $Mg^{++}$. The DNA cleavage activity of EcoRI endonuclease in inactivated by DFP, while the binding capacity of enzyme is not inactivated. Several lines of experimental evidence indicate that serine residue plays a very important catalytic role is EcoRI endonuclease. To generate peptide maps of the active serine residue region, the enzyme was labeled with $(^3H)$DFP and digested with trypsin. The amino acid composition of the labeled peptide corresponds to a tryptic fragment including residues 146-169 in the EcoRI endonuclease sequence.

제한 효소 EcoRl 의 활성에 관여하는 Serine 잔기 및 그 위치에 관한 연구

권세창,조영동 ( Se Chang Kwon,Young Dong Cho ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.2

EcoRl endonuclease is inactivated by diisopropylfluorophosphate (DFP), a serine residue modification reagent. The rate of inactivation is reduced by the substrate DNA and Mg^(++), The DNA cleavage activity of EcoRI endonuclease in inactivated by DFP, while the binding capacity of enzyme is not inactivated. Several lines of experimental evidence indicate that serine residue plays a very important catalytic role is EcoRI endonuclease. To generate peptide maps of the active serine residue region, the enzyme was labeled with (³H)DFP and digested with trypsin. The amino acid composition of the labeled peptide corresponds to a tryptic fragment including residues 146-169 in the EcoRI endonuclease sequence.

제한효소 Bam Hi 의 필수아미노산 잔기들과 그 역할에 관한 연구

조진만,권세창,조영동 ( Jin Man Cho,Se Chang Kwon,Young Dong Cho ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.4

To determine amino acid residues essential for the activity of BamHI endonuclease, we have used pyridoxal-5-phosphate(PLP), diethylpyrocarbonate(DEP), diisopropylfluorophosphate (DFP), and phenylglyoxal. Inactivation of BamHI endonuclease by PLP and DEP follows pseudo-first order kinetics. And the second order rate constants for the inactivation of the enzyme were 650 M^(-1) min^(-1) and 1125 M^(-1) min^(-1), respectively. Preincubation with group specific reagents for lysine, histidine, serine and arginine residues resulted in the loss of hydrolytic activity and DNA binding function. Furthermore, the presence of inactivation of the enzyme with increasing PLP and phenylglyoxal concentrations correlated very well with quantitative decrease in catalytic activity and binding capacity. However, the complete loss of the enzyme activity with increasing concentrations of DEP and DFP correlated not with decrease in binding capacity, but with decrease in catalytic activity. The cumulative results suggest that histidine and serine residues play a hydrolysis role for the phosphodiester bond, i.e., in the catalytic step, and lysine and arginine residues play a very important role as binding residues in BamHI endonuclease.

제한효소 Bam HI의 필수아미노산 잔기들과 그 역할 때 관한 연구

조진만,권세창,조영동,Cho, Jin-Man,Kwon, Se-Chang,Cho, Young-Dong 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.4

제한효소 BamHI에서 활성에 관여하는 필수적인 아미노산을 알기 위하여 PLP, DEP, DFP와 PG를 효소에 작용시켰다. 효소에 PLP와 DEP을 작용시킨 결과 비활성화 되었으며, 유사 일차반응이었다. 그리고 효소에 PLP와 DEP를 각각 작용시 이차반응속도는 각각 $650M^{-1}min^{-1}$와 $1125M^{-1}min^{-1}$였다. Lysine, histidine, serine과 arginine에 선택적으로 작용하는 PLP, DEP, DFP와 PG를 효소에 각각 반응시 절단능과 결합능의 감소가 일어났다. 효소는 기질에 의하여 상기한 화합물에 의한 비활성화를 억제할 수 있었다. 효소의 비활성화 정도는 각각 PLP와 PG의 농도에 비례하였고, 또한 절단능의 감소와 결합능력의 감소도 각각 정량적인 관계가 있었다. 반면에 DEP와 DFP 농도의 증가에 따른 효소활성의 감소는 절단능과 관계가 없고 결합능의 감소에 기인하였다. 이상과 같은 결과에 의하면 histidine과 serine은 phosphodiester 절단능과 관련이 있고 lysine과 arginine은 결합능에 관여할 것으로 사료된다. To determine amino acid residues essential for the activity of BamHI endonuclease, we have used pyridoxal-5-phosphate(PLP), diethylpyrocarbonate(DEP), diisopropylfluorophosphate (DFP), and phenylglyoxal. Inactivation of BamHI endonuclease by PLP and DEP follows pseudo-first order kinetics. And the second order rate constants for the inactivation of the enzyme were $650M^{-1}min^{-1}$ and $1125M^{-1}min^{-1}$, respectively. Preincubation with group specific reagents for lysine, histidine, serine and arginine residues resulted in the loss of hydrolytic activity and DNA binding function. Furthermore, the presence of inactivation of the enzyme with increasing PLP and phenylglyoxal concentrations correlated very well with quantitative decrease in catalytic activity and binding capacity. However, the complete loss of the enzyme activity with increasing concentrations of DEP and DFP correlated not with decrease in binding capacity, but with decrease in catalytic activity. The cumulative results suggest that histidine and serine residues playa hydrolysis role for the phosphodiester bond, i.e., in the catalytic step, and lysine and arginine residues playa very important role as binding residues in BamHI endonuclease.

The Role of Histidyl Residues in EcoRI Endonuclease

권세창,조진만,조영동,Kwon, Se-Chang,Cho, Jin-Man,Cho, Young-Dong 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.3

제한효소 Eco RI을 histidine residue에 반응하는 diethylpyrocarbonate(DEP)와 반응시킨 결과 효소 활성이 감소함을 알 수 있었다. 효소 활성이 90% 정도 감소할 때까지 반응은 pseudo-first order kinetics를 따랐고 DEP와 더 반응시키면 효소 활성이 완전히 없어졌다. DEP에 의한 효소의 비활성화는 기질인 pUC9 DNA에 의해 보호됨을 알 수 있었고 1개의 histidine residue가 효소 활성에 관여하는 것으로 나타났다. 그리고 DEP에 의해 비활성화된 효소에 과량의 hydroxylamine을 첨가하여 주면 재활성이 나타났다. 한편 histidine residue의 역할을 알아보고자 DEP가 제한효소 EcoRI의 기질 결합 능력에 미치는 영향을 조사하였다. 효소의 기질 결합 능력도 DEP 농도를 증가시킴에 따라 감소하였고 기질에 대한 보호 효과와 hydroxylamine에 의한 재활성화가 위의 효소 비활성화와 비슷하게 나타났다. 따라서 제한효소 EcoRI의 비활성화는 기질에 결함을 하지 못함으로써 나타난 것으로 생각된다. 이런 결과에 따르면 제한 효소 EcoRI의 활성에 histidine residue가 관여하며 이 residue는 기질에 대한 결합 기작에 관여하는 것으로 사료된다. Treatment of EcoRI endonuclease with the histidine modification reagent, diethylpyrocarbonate(DEP), resulted in enzyme inactivation. The reaction followed pseudo first-order kinetics until 90 to 95% of the enzyme had been inactivated. And prolonged incubation with DEP resulted in complete inactivation. When the modification reaction was carried out after preincubation with pUC9 DNA as substrate, the rate of inactivation was significantly decreased. The kinetics of inactivation indicate that the reaction of 1 molecule of DEP reagent is necessary for inactivation of each active site in EcoRI endonuclease. The inactivation of EcoRI endonuclease by DEP was partially reversed upon addition of excess hydroxy lamine. The effects of DEP on the binding ability of EcoRI endonuclease to its substrate, pUC9 DNA, were also investigated. When EcoRI endonuclease was preincubated with substrate DNA, the binding capacity was recovered significantly which suggests protection of the binding activity from DEP. Binding activity was also recovered with addition of hydroxylamine. The inhibition of activity, protection by DNA from DEP modification or reactivation by addition of hydroxylamine show similar tendencies to that of catalytic and binding properties. The cumulative results suggest that histidine residue plays a very important role as a binding residue in EcoRI endonuclease.

제한효소 Eco Rl의 Histidine Residue 역할에 관한 연구

권세창,조진만,조영동 ( Se Chang Kwon,Jin Man Cho,Young Dong Cho ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.3

Treatment of EcoRI endonuclease with the histidine modification reagent, diethylpyrocarbonate(DEP), resulted in enzyme inactivation. The reaction followed pseudo first order kinetics until 90 to 95% of the enzyme had been inactivated. And prolonged incubation with DEP resulted in complete inactivation. When the modification reaction was carried out after preincubation with pUC9 DNA as substrate, the rate of inactivation was significantly decreased. The kinetics of inactivation indicate that the reaction of 1 molecule of DEP reagent is necessary for inactivation of each active site in EcoRI endonuclease. The inactivation of EcoRl endonuclease by DEP was partially reversed upon addition of excess hydroxylamine. The effects of DEP on the binding ability of EcoRI endonuclease to its substrate, pUC9 DNA, were also investigated. When EcoRI endonuclease was preincubated with substrate DNA, the binding capacity was recovered significantly which suggests protection of the binding activity from DEP. Binding activity was also recovered with addition of hydroxylamine. The inhibition of activity, protection by DNA from DEP modification or reactivation by addition of hydroxylamine show similar tendencies to that of catalytic and binding properties. The cumulative results suggest that histidine residue plays a very important role as a binding residue in EcoRI endonuclease.

Purification and Characterization of XhoII Endonuclease from Xanthomonas holcicola

권세창,조진만,강정훈,김주훈,조영동,Kwon, Se-Chang,Cho, Jin-Man,Kang, Jung-Hoon,Kim, Joo-Hoon,Cho, Young-Dong 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.2

XhoII endonuclease를 Xanthomonas holcicola로부터 분리 하였으며 여러 group specific reagents를 사용하여 효소의 비활성화, 기질에 의한 비활성화 억제와 비활성화된 효소의 재활성화 등을 조사하여 효소 활성에 관여하는 amino acid residue를 조사하였다. XhoII endonuclease의 분자량은 gel filteration을 통해 76,000으로 나타났고 이 효소의 최적온도는 $37^{\circ}C$이고 pH7.5에서 8.0사이에는 활성도에 변화을 주지 않는 최적 pH임이 밝혀졌다. XhoII endonuclease에 group specific reagents인 phenylglyoxal(arginine), PLP와 O-phthalaldehyde(lysine), DTNB와 NEM(cysteine) 그리고 DFP (serine)을 적용시킨 결과 효소활성이 억제되었다. DTNB와 NEM, phenylglyoxal 그리고 DFP에 의한 효소활성 억제에 대한 기질인 pUC9 DNA가 보호 효과를 보였으며 특히 DTNB와 NEM에 의한 효소의 비활성화는 ${\beta}$-MSH에 의해 활성이 회 복되었다. 이런 결과로 XhoII endonuclease활성에 arginne, serine 그리고 cysteine residue가 관여할 것이라 생각되며 특히 cysteine residue가 중요한 역할을 하고 있을 것이라 사료된다. XhoII endonuclease was purified from Xanthomonas holcicola by streptomycin sulfate fractionation, phosphocellulose, heparin agarose, Sephadex G-100, DNA-cellulose and DEAE-sephacel column chromatography. The molecular weight of XhoII endonuclease was 76,000 measured by Sephadex G-100 and G-200 column chromatography. 22,000 units of XhoII endonuclease was obtained from 100 g of cell paste. XhoII endonuclease has an optimum temperature of $37^{\circ}C$ with pH 7.5 to 8.0. XhoII endonuclease activity was remarkably inhibited above a NaCl concentration of 150 mM. XhoII endonuclease activity was also severely inhibited above 0.5 mM NEM and 1 mM DTNB, the thiol group modification reagents, and 3 mM phenylglyoxal, the arginine modification reagent. But XhoII endonuclease from inactivation by these reagents was protected by substrate DNA. So there is a possibility that the thiol group and arginine residue play an important role in the XhoII endonuclease activity.

Xanthomonas holcicola 에서 Xhaoll endonuclease 분리 및 성질

권세창,조진만,강정훈,조영동 ( Se Chang Kwon,Jin Man Cho,Jung Hoon Kang,Joo Hoon Kim,Young Dong Cho ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.2

XhoII endonuclease was purified from Xanthomonas holcicola by streptomycin sulfate fractionation, phosphocellulose, heparin agarose, Sephadex G-100, DNA-cellulose and DEAE-sephacel column chromatography. The molecular weight of XhoII endonuclease was 76,000 measured by Sephadex G-100 and G-200 column chromatography. 22,000 units of XhoII endonuclease was obtained from 100 g of cell paste. XhoII endonuclease has an optimum temperature of 37℃ with pH 7.5 to 8.0. XhoII endonuclease activity was remarkably inhibited above a NaCI concentration of 150 mM. XhoII endonuclease activity was also severely inhibited above 0.5 mM NEM and 1 mM DTNB, the thiol group modification reagents, and 3 mM phenylglyoxal, the arginine modification reagent. But XhoII endonuclease from inactivation by these reagents was protected by substrate DNA. So there is a possibility that the thiol group and arginine residue play an important role in the XhoII endonuclease activity.