The enzyme responsible for the direct phosphorylation of pyruvate during gluconeogenesis in Acetobacter xylinum has been purified 46-fold from ultrasonic extracts and freed from interfering enzyme activities. The enzyme was shown to catalyze the reversible Mg2+ ion-dependent conversion of equimolar amounts of pyruvate, adenosine triphosphate (ATP), and orthophosphate (Pi) into phosphoenolpyruvate (PEP), adenosine monophosphate (AMP), and pyrophosphate (PP). The optimal pH for PEP synthesis was pH 8.2; for the reversal it was pH 6.5. The ratio between the initial rates of the reaction in the forward and reverse directions was 5.1 at pH 8.2 and 0.45 at pH 6.5. The apparent Km values of the components of the system in the forward reaction were: pyruvate, 0.2 mm; ATP, 0.4 mm; Pi, 0.8 mm; Mg2+, 2.2 mm; and for the reverse reaction: PEP, 0.1 mm; AMP, 1.6 μm; PP, 0.067 mm; Mg2+, 0.87 mm. PEP formation was inhibited by AMP and PP. The inhibition by AMP was competitive with regard to ATP (Ki = 0.2 mm). The reverse reaction was inhibited competitively by ATP and noncompetitively by pyruvate. The enzyme was strongly inhibited by p-hydroxymercuribenzoate. The inhibition was reversed by dithiothreitol and glutathione. The properties of the enzyme are discussed in relation to the regulation of the opposing enzymatic activities involved in the interconversion of PEP and pyruvate in A. xylinum.
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机译：木糖醋杆菌糖异生过程中负责丙酮酸直接磷酸化的酶已从超声提取物中纯化了46倍，并且没有干扰酶的活性。该酶被证明可催化等摩尔量的丙酮酸，三磷酸腺苷（ATP）和正磷酸（P i ）等摩尔量的Mg 2 + 离子依赖性转化为磷酸烯醇丙酮酸（ PEP），单磷酸腺苷（AMP）和焦磷酸（PP）。 PEP合成的最佳 p H为 p H 8.2;逆转是 p H 6.5。在 p H 8.2时，在正向和反向反应的初始速率之间的比率为5.1，在 p H 6.5时为0.45。在正向反应中，系统各组分的表观 K m 值为：丙酮酸0.2 mm; ATP，0.4毫米; P i ，0.8毫米; Mg 2 + ，2.2毫米;对于逆反应：PEP为0.1mm; AMP，1.6微米; PP，0.067毫米; Mg 2 + ，0.87毫米。 PEP的形成被AMP和PP抑制。 AMP对ATP的抑制作用是竞争性的（ K i = 0.2 mm）。反向反应被ATP竞争性抑制，而丙酮酸则非竞争性地抑制。该酶被 p -羟基巯基苯甲酸强烈抑制。二硫苏糖醇和谷胱甘肽逆转了抑制作用。讨论了酶的性质与在emA中PEP和丙酮酸的相互转化中涉及的相反酶活性的调节有关。木糖。
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