In Vitro Translation of Glucose-6-Phosphate Dehydrogenase Isozyme mRNA and In Vivo Isotope Labeling of the Enzyme from Peking Duck

이상철,김유삼,Lee, Sang-Chul,Kim, Yu-Sam 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.3

오리의 uropygial gland와 간에서 $poly(A)^+mRNA$를 분리하여 그들의 in vitro translation mixture로부터 pI 6.6인 glucose-6-phosphate dehydorgenase를 분리 하고 in vivo isotope labeling된 같은 효소를 분리하여 SDS polyacrylamide disc gel electrophoresis를 통하여 비교하였다. 그 결과 in vitro translation mixture로 부터 분리한 효소와 in vivo labeling된 효소는 그것이 uropygial gland의 것이거나 간의 것이거나 모두가 uropygial gland에서 직접 정제한 효소와 같은 크기임을 알 수 있었다. 이러한 사실은 uropygial gland에 존재하는 pI 6.6인 glucose-6-phosphate dehydrogenase가 갖는 효소적 특성은 소수의 아미노산의 차이에 기인하는 것으로 예측된다. One glucose-6-phosphate dehydrogenase with pI of 6.6 was isolated from in vitro translation mixture and was compared with in vivo isotopically labeled enzyme by SDS polyacrylamide gel electrophoresis. The molecular size of the enzymes, isolated by antibody prepared against the purified enzyme and protein A agarose, are very similar to each other. This result suggests that the specific catalytic properties of the enzyme from the uropygial gland may depend on the minor difference in amino acid sequence.

오리의 Urpygial Gland 에 있는 Glucose - 6 - phosphate Dehydrogenase 의 활성에 관여하는 Essential Cysteine 과 Arginine residues

이상철,김유삼 ( Sang Chul Lee,Yu Sam Kim ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.2

Glucose-6-phosphate dehydrogenase from the uropygial gland of duck was purified in an electrophoretically homogenous form and its essential amino acid for catalysis was investigated. Three -SH group directed reagents, p-chloromercuriphenylsulfonic acid, 5,5`-dithio-bis-(2-nitrobenzoic acid) and N-ethylmaleimide, inhibited the enzyme activity, and arginine directed reagent, phenylglyoxal, also inhibited the enzyme activity. The inhibition of the enzyme by these reagents was protected by addition of NADP+ whereas it was not affected by that of glucose-6-phosephate indicating that essential -SH and guanidino group are located on the NADP^+ binding region of active site. Mg^(++) promoted the inhibition of the enzyme by the -SH and guanidine .group directed reagents. But the promotion of the inhibition was considerably recovered in the presence of EDTA. This results suggested that divalent metal cation may affect the conformational change toward the susceptible one against modifying reagents.

Identification of Essential Cysteine and Arginine Residues in Glucose-6-phosphate Dehydrogenase from the Uropygial Gland of Duck

이상철,김유삼,Lee, Sang-Chul,Kim, Yu-Sam 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.2

오리의 uropygial gland로 부터 pI 6.6인 glucose-6-phosphate dehydorgenase를 순수하게 정제하여 효소활성과 기질 및 보조효소와의 결합에 관여하는 아미노산에 대해 조사하였다. Cysteine의 -SH기에 특이하게 반응하는 p-chloromercuriphenylsulfonic acid, 5,5'-dithio-bis-(2-nitrobenzoic acid), N-ethylmaleimide와 arginine의 guanidino기에 특이하게 반응하는 phenylglyoxal 등이 효소의 활성을 억제한다는 사실로 보아 효소의 active site나 그 근처에 cysteine과 arginine이 존재하는 것으로 예상된다. 또한 glucose-6-phosphate가 존재할 때 이 reagent들에 의한 효소의 활성억제에 아무런 영향이 없었으나 $NADP^+$존재하에서는 효소의 활성억제 현상이 크게 감소한다는 사실로 미루어 cysteme과 arginine이 $NADP^+$와 효소가 결합하는 위치냐 또는 그 부근에 있는 것으로 판단된다. $Mg^{++}$이 존재할 때 p-chloromercuriphenylsulfonic acid, 5,5'-dithio-bis(2-nitrobenzoic acid)와 phenylglyoxal에 의한 효소의 활성 억제가 더 크게 나타났는데, 이러한 사실은 EDTA와 $Mg^{++}$이 공존할 때 이 reagent에 의한 활성 억제가 EDTA가 존재하지 않을 때 보다 감소하였다는 것으로 보아 $Mg^{++}$이 이 reagent에 의한 효소의 활성 억제를 증가시킨다는 사실을 확인하였다. Glucose-6-phosphate dehydrogenase from the uropygial gland of duck was purified in an electrophoretically homogenous form and its essential amino acid for catalysis was investigated. Three -SH group directed reagents, p-chloromercuriphenylsulfonic acid, 5,5'-dithio-bis-(2-nitrobenzoic acid) and N-ethylmaleimide, inhibited the enzyme activituy, and arginine directed reagent, phenylglyoxal, also inhibited the enzyme activity. The inhibition of the enzyme by these reagents was protected by addition of $NADP^+$ whereas it was not affected by that of glucose-6-phosephate indicating that essential -SH and guanidino group are located on the $NADP^+$ binding region of active site. $Mg^{++}$ promoted the inhibition of the enzyme by the -SH and guanidino group directed reagents. But the promotion of the inhibition was considerably recovered in the presence of EDTA. This results suggested that divalent metal cation may affect the conformational change toward the susceptible one against modifying reagents.

Rhizobium trifolii Malonyl - CoA Synthetase 의 촉매반응에 관여하는 아미노산 Residue 의 화학변형

이상철,김유삼 ( Sang Chul Lee,Yu Sam Kim ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.3

Malonyl-CoA synthetase from Rhizobium trifolii was inactivated by 2,3-butanedione and diethylpyrocarbonate (DEP). In each case, inactivation followed pseudo first-order kinetics. The second-order rate constant for the inactivation by 2,3-butanedione was 17.4M^(-1)·min^(-1) at pH 8.0, 30℃. DEP rapidly inactivated the enzyme with the second-order rate constant, 780 M^(-1)·min^(-1) at pH 7.1, 30℃. The reaction order of inactivation with respect to reagents were 1.06 and 1.03 respectively. The substrate, ATP, afforded the protection against the inactivation of the enzyme caused by the reagent, respectively. These results indicate that arginine and histidyl residues essential for the enzyme activity are located at or near the ATP binding region of the active site.

Rhizobium trifolii Malonyl-CoA Synthetase의 촉매반응에 관여하는 아미노산 Residue의 화학변형

이상철,김유삼,Lee, Sang-Chul,Kim, Yu-Sam 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.3

Rhizobium tnifolii에서 정제한 malonyl-CoA synthetase가 2,3-butanedione과 diethylpyro-carbonate(DEP)에 의해 불활성화 되었다. 각 불활성화 반응은 pseudo first-order kinetics으로 나타났다. 2,3-Butanedion에 의한 효소의 불활성화 반응의 2차 속도상수는 pH 8.0, $30^{\circ}C$에서 $17.4M^{-1}{\cdot}min^{-1}$이었다. DEP에 의해 효소가 빠르게 불활성화되었으며 반응의 2차속도상수는 pH 7.1, $30^{\circ}C$ 에서 $780M^{-1}{\cdot}min^{-1}$이었다. 각 시약과 반응차수는 1.06과 1.03이였다. ATP가 위의 2가지 시약에 의한 효소의 불활성화를 저해하였다. 이러한 결과들은 malonyl-CoA synthetase의 ATP 결합부위나 그 주위에 효소활성에 필수적인 arginine residue와 histidyl residue가 존재함을 시사한다. Malonyl-CoA synthetase from Rhizobium trifolii was inactivated by 2,3-butanedione and diethylpyrocarbonate (DEP). In each case, inactivation followed pseudo first-order kinetics. The second-order rate constant for the inactivation by 2,3-butanedione was $17.4M^{-1}{\cdot}min^{-1}$ at pH 8.0, $30^{\circ}C$. DEP rapidly inactivated the enzyme with the second-order rate constant, $780M^{-1}{\cdot}min^{-1}$ at pH 7.1, $30^{\circ}C$. The reaction order of inactivation with respect to reagents were 1.06 and 1.03 respectively. The substrate, ATP, afforded the protection against the inactivation of the enzyme caused by the reagent, respectively. These results indicate that arginine and histidyl residues essential for the enzyme activity are located at or near the ATP binding region of the active site.

Pyridoxal-5'-Phosphate에 의한 Rhizobium trifolii Malonyl-CoA Synthetase의 화학변형

이상철,김유삼,Lee, Sang-Chul,Kim, Yu-Sam 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.3

Pyridoxal-5’-phosphate가 Rhizobium tifolii에서 정제한 malonyl-CoA synthetase를 가역적으로 불활성화 하였다. 반응의 2차 속도상수는 pH 7.0, $30^{\circ}C$에서 $2975 M^{-1}{\cdot}min^{-1}$이었다. PLP에 의해 불활성화된 효소는 완충용액으로 희석하였을 때 가역적으로 활성을 회복하였으나, $NaBH_4$로 환원 후 희석한 효소는 활성을 회복하지 못하였다. PLP와 반웅 후 $NaBH_4$로 환원한 효소액은 325 nm에서 톡징적인 Schiff base의 홉광도 변화를 보였다. ATP가 PLP에 의한 효소의 불활성화를 저해하였다. 이러한 결과들은 malonyl-CoA synthetase의 ATP 결합부위나 그 주위에 효소활성에 필수적인 amine group이 존재함을 시사한다. Pyridoxal-5'-phosphate inactivated malonyl-CoA synthetase from Rhizobium trifolii reversibly. The second-order rate constant, $K_i$ was $2975 M^{-1}{\cdot}min^{-1} $ at pH 7.0, $30^{\circ}C$. Inactivation of malonyl-CoA synthetase by PLP was reversed by dilution but not after reduction with $NaBH_4$. The spectra of malonyl-CoA synthetase, inactvated by PLP and reduced by $NaBH_4$, showed a characteristic Schiff base absorption change at 325 nm. ATP protected the enzyme from inactivation by PLP. These studies indicated that an essential amine is located at or near ATP binding region of the enzyme active site.

Pyridoxal - 5 ' - Phosphate 에 의한 Rhizobium trifolii Malonyl - CoA Synthetase 의 화학변형

이상철,김유삼 ( Sang Chul Lee,Yu Sam Kim ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.3

Pyridoxal-5`-phosphate inactivated malonyl-CoA synthetase from Rhizobium trifolii reversibly. The second-order rate constant, K_i was 2975 M^(-1) · min^(-1) at pH 7.0, 30℃. Inactivation of malonyl-CoA synthetase by PLP was reversed by dilution but not after reduction with NaBH₄. The spectra of malonyl-CoA synthetase, inactvated by PLP and reduced by NaBH₄, showed a characteristic Schiff base absorption change at 325 nm. ATP protected the enzyme from inactivation by PLP. These studies indicated that an essential amine is located at or near ATP binding region of the enzyme active site.

오리에 있는 Glucose - 6 - Phosphate Dehydrogenase 한 Isozyme 의 in Vitro Translation 및 그 효소의 In vivo Isotope Labeling

이상철,김유삼 생화학분자생물학회 1991 BMB Reports Vol.18 No.3

One glucose-6-phosphate dehydrogenase with pI of 6.6 was isolated from in vitro translation mixture and was compared with in vivo isotopically labeled enzyme by SDS polyacrylamide gel electrophoresis. The molecular size of the enzymes, isolated by antibody prepared against the purified enzyme and protein A agarose, are very similar to each other. This result suggests that the specific catalytic properties of the enzyme from the uropygial gland may depend on the minor difference in amino acid sequence.