Mechanistic Studies of Enzymes involved in the Reversible Deamination and the Aldol Condensation

Abstract

PART I. 가역적 탈아민화 반응에서 Hafnia alvei Aspartase 의 기능적 변형과 반응 메카니즘 Hafnia alvei Aspartase는 DEAE-cellulose와 Red A-agarose 그리고 Sepharose 6B chromatography 조합을 통해 균질하게 정제되었다. 정제된 효소는 denatured SDS-polyacrylamide gel electrophoresis를 통해 동질의 균질성이 나타났고, 분자량 55,000Da의 동일한 소단위체로 구성된 4-단위체 단백질로 학인되었다. 효소반응을 위한 최적의 pH는 8.5였고 최대활성도를 위한 최적의 온도는 45°C였다. 효소는 염기성 pH 상태에서는 Mg^(2+), Mn^(2+) 등의 2가의 금속이온을 절대적으로 필요로 하지만 Zn^(2+), Ca^(2+) 등과 같은 다른 종류의 2가 금속이온 존재하에서는 비활성되었다. Circular dichroism spectropolarimetry를 이용한 정제된 효소의 Helical content는 61%로 산출되었다. H. alvei Aspartase의 활성도는 무수아세트산 처리에 의해 점차 증가하여 본래 효소의 7.5배 가까이 활성이 증가하였다. 아세틸화된 Aspartase의 활성도가 본래 효소보다 증가하였다는 것은 기질과 효소 사이의 협동성이 증가하였다는 것을 나타낸다. 본래 효소의 최적 온도는 45°C였고 아세틸화된 효소는 40°C로 이동되었다. 아세틸화된 효소의 pH와 활성도 관련성 역시, 본래 효소와는 다르게 나타났다. 아세틸화된 효소의 Initial velocity patterns는 축의 왼쪽에서 교차하는데, 이것은 Rapid equilibrium ordered mechanism보다는 Sequential kinetic mechanism을 나타낸다. 본래 효소의 Aspartate에 대한 Reciprocal plots은 곡선이지만, 아세틸화된 효소는 직선으로 나타나고 이것은 Michaelis-Menten kinetics을 의미한다. 아세틸화된 효소의 Helical content는 본래 효소(63%)보다 9% 감소하였다. 생체 조건 밖에서 H. alvei Aspartase를 0.2-4.0M 범위에서 Gu-HCl으로 변성시키면 1.0M Gu-HCl까지는 활성도가 점점 감소하다가 1.0M Gu-HCl 이상에서는 완전히 활성을 잃었다. 그러나, 약 100배 희석하면 1.0M Gu- HCl까지는 100% 재활성되었고 1.0M Gu-HCl 이상에서는 약 40% 재활성되었다. 이러한 결과는 Tetramer에서 Dimmer로의 Dissociation process가 가역적이라는 것을 의미한다. 반대로, 1.0M Gu-HCl이상에서 재활성이 40%라는 것은 Monomer 로의 Dissociation process가 가역적이지 않다는 것을 의미한다. pH와 온도 같은 요인들은 재활성 속도와 범위에 영향을 미친다. 재활성에 최적의 pH와 온도는 pH7.0 그리고 25°C였다. 4°C에서는 변성된 효소를 희석하여도 활성이 나타나지 않았으나 25°C로 온도를 올리게 되면 활성 속도가 빠르게 증가함이 관찰되었다. 그러나, 37°C 이상으로 온도를 올리게 되면 효소는 활성을 완전히 잃게 되었고, 이러한 실험 결과는 재활성 과정에서 어떤 온도-의존 과정이 속도-결정 단계를 포함하고 있다는 것을 의미한다. 금속이 없는 상태에서 아민화반응 방향으로의 H. alvei Aspartase의 반응 메커니즘이 결정되었다. Initial velocity patterns는 몇 개의 고정된 NH₄^(+) 농도에서 Fumarate의 농도를 변화시키면서 확인하였고, pH7.0 상태에서 축의 왼쪽에서 교차하는데 이것은 반응 메커니즘이 Fumarate에 의해 기질 저해가 관찰되는 Sequential mechanism이란 것을 의미한다. 몇 개의 고정된 Succinate 농도에서 NH₄^(+)의 농도를 변화시키면서 확인한 Dead-end inhibition patterns는 축의 왼쪽에서 교차하였다. 이러한 결과는 효소와 결합하는 NH₄^(+)와 Fumarate의 순서가 무작위적이라는 것과 일치한다. Haldane relationship을 통해 pH7.0에서 산출된 Equilibrium constant, Keq는 1.18 ´ 10^(-3)M인데, 이것은 평형에서 반응에 관여하는 농도들을 통해 직접 결정하는 방법으로 얻어진 Equilibrium constant 값 6.0 ± 0.2 ´ 10^(-3) M과 거의 일치하는 값이다. PART II. 초고온성 원시균인 Methanococcus Jannaschii FucA 의 기능적 변형과 반응 메카니즘 DHAP와 L-lactaldehyde로부터 Fuculose-1-phosphate를 생성하는 알돌 축합 반응을 촉매하는 Methanococcus Jannaschii FucA는 DEP에 의해 비활성화되는 효 소이다. 비활성은 두 가지 상을 나타내는 효소와 DEP의 유사 일차 반응이었다. pH6.0과 25°C에서 유사 이차 속도 상수는 120M^(-1)min^(-1)이었다. 비활성화 속도는 pH에 의존적이었고, pK_(a) 값 5.7을 가지는 아미노산 잔기를 포함하는 pH 의존적 비활성화를 나타냈다. FucA는 DHAP에 의해 DEP에 의한 비활성화를 막을 수 있 었는데, 이는 FucA의 활성화자리에 Histidine 잔기들이 위치하고 있다는 것을 의 미한다. L-lactaldehyde에 대한 선택적 기질로써 DL-glyceraldehyde는 FucA의 비 활성화 방어에 대하여 특이적인 결과가 나타나지 않았다. M. Jannaschii FucA의 축합반응과 가수분해반을을 포함하는 가역적 알돌 축 합 반응에서의 전체적인 반응 메카니즘을 밝히기 위하여 Initial velocity patterns, Kinetic parameters, Dead-end inhibition patterns, Product inhibition patterns 그리고 Equilibrium constant 등을 확인하였다. Ketone과 Aldehyde로부터 D-psicose-1- phosphate와 L-tagatose-1-phosphate을 생성하는 알돌 축합 반응에서 FucA은 기질 로써 DHAP와 DL-glyceraldehyde을 활용하여 연구가 이루어져 왔다. 반응속도는 FucA의 직선 함수였고, 기질로써 DHAP와 DL-glyceraldehyde에 대한 K_(m) 값은 각각 9.0998 × 10^(-2)mM과 7.4301 × 10^(-1)mM 이었다. 몇 개의 고정된 DHAP 농도에 서 의 농도를 변화시키면서 확인한 Initial velocity patterns는 축의 왼쪽에서 교차 하는데, 이것은 효소의 반응 메커니즘이 sequential 메커니즘이라는 것을 의미한다. Dead-end inhibition은 DHAP와 DL-glyceraldehyde의 기질 유사체로써 TMPA와 DL-threose을 사용하여 확인되었는데, TMPA은 DHAP와 DL-glyceraldehyde에 대 하여 competitive 저해가 나타났고, DL-threose은 DHAP에 대해서는 uncompetitive, 그리고 DL-glyceraldehyde 에 대해서는 competitive 저해가 나타났 다. 이러한 Dead-end inhibition patterns는 FucA가 ordered sequential 메커니즘이 라는 것을 의미한다. 생성물인 D-psicose-1-phosphate와 L-tagatose-1-phosphate는 “The methods of phosphate-barium salt with ion-exchange resin (Bednarski et al., 1989)”를 이용하여 얻었고 Product inhibition patterns는 DHAP에 대해서는 noncompetitive, DL-glyceraldehyde에 대해서는 competitive 저해를 나타냈다. 이 가역반응에 대하여 Haldane relationship을 통해 얻은 평형상수는 18.3344 × 10-3M 이었고, 고정된 [DL-glyceraldehyde] 농도에서 [DHAP]/[product]의 비율 변화와 평형에 도달한 후 DHAP의 농도 차이에 의해 평형상수를 직접 결정하는 방법을 사용하여 얻은 평형상수는 8.3093 × 10^(-3)M이었고, 이것은 (13)^C NMR을 이용하여 얻 은 평형상수 15.6250 × 10^(-3)M과 거의 일치되는 값이었다.; PART I. Functional Modification and Kinetic Mechanism of Aspartase from Hafnia alvei in the Reversible Deamination. Aspartase from Hafnia alvei was purified to homogeneity by a combination of DEAE-cellulose, Red A-agarose and Sepharose 6B chromatography. The purified enzyme appeared homogeneous on denatured SDS-polyacrylamide gel electrophoresis and was a tetrameric protein composed of identical subunits with a molecular weight of 55,000Da. The optimum pH for the enzymatic reaction was 8.5 and the optimum temperature for maximal activity was 45?C. The enzyme has an absolute requirement of divalent metal ions (Mg2+, Mn2+) at the alkaline pH. The enzyme, however, was inactivated in the presence of other divalent cations such as Zn2+, Ca2+. The helical content of the purified enzyme was estimated by CD spectropolarimetry to be 61%. The activity of Hafnia alvei aspartase with acetic anhydride treatment gradually increased and reached 7.5-fold that of the native one. The activity of the acetylated aspartase was a little higher than that of the native enzyme, indicating that the cooperativity between a substrate and enzyme is increased. The optimal temperature of the native asparatse was 45?C, and that of the acetylated enzyme shifted to 40?C. The pH vs. the activity profile of the acetylated asparatse was also different from that of the native enzyme. The initial velocity pattern of the acetylated aspartase intersects to the left of the ordinate, indicating a sequential kinetic mechanism other than a rapid equilibrium ordered one. The reciprocal plots for aspartate of the native aspartase were curved, but those of the acetylated aspartase were linear, indicating the Michaelis-Menten kinetics. The helical content (54%) of the acetylated aspartase was rather decreased to 7% than that (61%) of the native one. When aspartase from Hafnia alvei was denaturated in vitro in the range of 0.2M to 4.0M Gu-HCl, activity was slowly decreased up to 1.0M Gu-HCl and totally lost above 1.0M Gu-HCl. It was reactivated about 100% by 100-fold dilution up to 1.0M Gu-HCl and reactivated about 40% above 1.0M Gu-HCl. These results indicate that the dissociation process from tetramer to dimmer is reversible. In contrast, the renaturation yield was 40% when the enzyme was diluted from more than 1M Gu-HCl, indicating that the process of dissociation into monomer is not reversible. The factors such as temperature and pH exhibited the influence on the rate and extent of the reactivation. The reactivation was best at pH7.0 and 25?C. Upon dilution of the denatured enzyme at 4?C, the reactivation of enzyme was not detected, up to 3hour. Upon the temperature shift up to 25?C, a rapid increase in the rate of reactivation was observed. But up to 1hour, the reactivation of enzyme was not observed because enzyme has need of time to remove the Gu-HCl by dilution. However, upon the temperature shift up to 37?C, the enzyme was totally lost the activity, indicating that some temperature-dependent process is involved as a rate-determining step in the reactivation process. The kinetic mechanism of Hafnia alvei asparatse in the amination direction has been determined in the absence of metal. The initial velocity patterns obtained by varying the concentration of fumarate at several fixed concentrations of NH4+, show an intersection on the left of the ordinate at pH7.0, indicating that the kinetic mechanism is a sequential mechanism in which substrate inhibition by fumarate is observed. The dead-end inhibition patterns by varying the concentration of NH4+ at several fixed concentration of succinate show an intersection on the left of the ordinate. These data are consistent with random addition of NH4+, or fumarate. The Haldane relationship gives a Keq of 1.18 ? 10-3M at pH7.0, which is in agreement with the value obtained from the direct determination of reaction concentrations at equilibrium (6.0 ? 0.2 ? 10-3M). PART II. Functional Modification and Kinetic Mechanism of Fuculose-1-phosphate Aldolase from Methanococcus jannaschii, Hyperthermophilic Archaea. When the activity of the purified FucA from Methanococcus jannaschii was measured at different pH, the highest activity was in the range of pH7.5 to 9.0. The FucA showed at least 30% of activity at one unit below pH7.0 or over pH9.0. The optimal temperature of the FucA was measured by determining the conversion rate of DHAP at different temperatures. The removal rate of DHAP increased according to the temperature. However, DHAP was also decomposed at high temperatures. Since the rate of thermal decomposition of DHAP under 37?C was less than 10%, this temperature was used for further characterization. The enzyme FucA from M. jannaschii that catalyzes the aldol condensation of DHAP and L-lactaldehyde to give fuculose-1-phosphate was inactivated by DEP. The inactivation was pseudo first-order in the enzyme and DEP, which was biphasic. A pseudo second-order rate constant of 120M-1min-1 was obtained at pH6.0 and 25?C. The rate of inactivation was dependent on the pH, and the pH inactivation data implied the involvement of an amino acid residue with a pKa value of 5.7. FucA was protected against DEP inactivation by DHAP, indicating that the histidine residues were located at the active site of FucA. DL-glyceraldehyde as an alternative substrate to L-lactaldehyde showed no specific protection for FucA. In order to define the overall kinetic mechanism of M. jannaschii FucA and also to elaborate the kinetic mechanism in the reversible aldol condensation reaction, condensation and hydrolysis reaction, we have determined its initial velocity patterns, kinetic parameters, dead-end inhibition patterns, product inhibition patterns and equilibrium constant. The condensation reaction of the FucA, aldol condensation of a ketone and an aldehyde to produce D-psicose-1-phosphate and L-tagatose-1-phosphate, has been studied by using DHAP and DL-glyceraldehyde as the substrates. Velocity was linear function of FucA and the Km values of DHAP and DL-glyceraldehyde were 9.0998 ? 10-2mM and 7.4301 ? 10-1mM, respectively. A determination of the initial velocity varying the concentration of DL-glyceraldehyde at different fixed levels of DHAP gave an intersection on the left of the ordinate, indicating that the kinetic mechanism of enzyme is a sequential. Dead-end inhibitions by TMPA and DL-threose as a substrate analogue of DHAP and DL-glyceraldehyde have also determined. Dead-end inhibition by TMPA was competitive with respect to DHAP and DL-glyceraldehyde. Dead-end inhibition by DL-threose was uncompetitive and competitive with respect to DHAP and DL-glyceraldehyde, respectively. These dead-end inhibition patterns indicate that the kinetic mechanism of FucA is an ordered sequential mechanism. Products, D-psicose-1-phosphate and L-tagatose-1-phosphate, were obtained by using the methods of phosphate-barium salt with ion-exchange resin. Inhibition patterns obtained by using the product were noncompetitive vs. DHAP and competitive DL-glyceraldehyde. Equilibrium constant for this reversible reaction obtained by using the Haldane relationship is 18.3344 ? 10-3M and by using the method of direct determination of equilibrium constant with varying the [DHAP]/[product] ratio at a fixed [DL-glyceraldehyde] and measuring the change in DHAP concentration after equilibrium is reached is 8.3093 ? 10-3M, in good agreement with the determined Keq values obtained from 13C NMR (15.6250 ? 10-3M).