Malonate를 유일한 탄소 및 에너지원으로 이용하는 배지에서 Acinetobacter calcoaceticus를 배양했을 때 malate synthase가 유도된다는 사실을 확인하였다. 이 미생물로부터 DEAE-Sephacel ion exchange chromatography, oxalate-Sepharose 4B affinity chromatography, hydroxyapatite chromatography를 이용하여 malate synthase를 분리 정제하였고, disc polyacrylamide gel electrophoresis를 수행한 결과 하나의 단백질 띠로 나타났다. 그 최적 pH는 8.0이였으며, 정제한 malate synthase가 관여하는 반응은 기질인 glyoxylate나 acetyl CoA에 대해 전형적인 Michaelis-Menten kinetics에 부합했다 Lineweaver-Burk plot을 이용하여 $K_m$과 $V_{max}$를 정한 결과 acetyl CoA에 대해서는 $1.5{ imes}10^{-5};M$과 $1;{mu}mol/min/mg$ protein이였고 glyoxylate에 대해서는 $3.5{ imes}10^{-5};M$과 $0.8;{mu}mol/min/mg$ protein 이었다. Oxalate와 glyoxylate는 competitive inhibitor였으며 $K_i$ 값은 각각 $4{ imes}10^{-5};M$, $1.4{ imes}10^{-4};M$이었다. Pyruvate, succinate, malonate 그리고 tartrate도 효소활성을 억제하였다. Pseudomonas fluorescens의 malate synthase를 항원으로 하여 만들어진 항체는 Acinetobacter calcoaceticus 의 malate synthase활성을 저해 하지 않았다. 이러한 사실로 미루어 분류상 유사한 두 미생물의 malate synthase는 면역학적으로 많은 차이가 있음을 알았다.
Malate synthase (EC 4.1.3.2) was induced in Acinetobacter calcoaceticus grown on malonate as a sole carbon source. The enzyme was purified by the combined methods of DEAE-Sephacel anion exchange chromatography, oxalate Sepharose 4B affinity chromatography and hydroxyapatite chromatography in an electrophoretically homogeneous form. The molecular size of the enzyme was estimated to be 75,000 dalton consisted of a single polypeptide. Optimum pH for the enzyme was about 8.0. The rate of acetyl group incorporation on variable concentraions of the substrate into malate was well fitted to a typical Michaelis-Menten kinetics. From the Lineweaver-Burk plot, $K_m$ and $V_{max}$ for acetyl-CoA were $1.5{ imes}10^{-5};M$ and $1;{mu}mol/min/mg$ respectively and those for glyoxylate were $3.5{ imes}10^{-5};M$ and $0.8;{mu}mol/min/mg$ respectively. The activity of this enzyme was inhibited by oxalate with $K_i$, $4{ imes}10^{-5};M$ and by glycolate with $K_i$, $1.4{ imes}10^{-4};M$ competitively. Pyruvate, succinate, malonate and tartrate also inhibited the enzyme activity. Antibody prepared against malate synthase from Pseudomonas fluorescens did not cross-react with the enzyme from Acinetobacter, suggesting that the enzyme isolated from two different bacteria are immunologocally different.
