ROLE OF LYSINE-183 IN D-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASES - PROPERTIES OF N-ACETYLATED YEAST, STURGEON MUSCLE AND RABBIT MUSCLE ENZYMES
ROLE OF LYSINE-183 IN D-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASES - PROPERTIES OF N-ACETYLATED YEAST, STURGEON MUSCLE AND RABBIT MUSCLE ENZYMES
复制标题
DOI：
10.1111/j.1432-1033.1978.tb12074.x
复制
发表时间：
1978-01-01
期刊：
影响因子：
--
通讯作者：
PUDLES, J
中科院分区：
文献类型：
--
作者：
FOUCAULT, G;NAKANO, M;PUDLES, J
兔肌肉、鲟鱼肌肉和酵母甘油醛-3-磷酸脱氢酶(EC 1.2.1.12)赖氨酸-183的乙酰化反应(EC 1.2.1.12)，由S在pH 8.5时完成。N-乙酰转移遵循一级反应过程。本质上，4个赖氨酸残基是修饰/酶四聚体。在3种底物(NAD+、3-甘油醛-3-磷酸和砷酸盐)饱和条件下，N-乙酰化的兔酶和鲟鱼肌酶的Vmax分别是天然酶的90%和80%，而N-乙酰化的酵母酶仅为20%。3种底物的Km值不受两种肌酶N-乙酰化的显著影响。然而，N-乙酰化酵母酶只对NAD+产生更高的Km。在Racker谱带进行分光光度滴定，结果表明，3种N-乙酰化的酶均能与4个NAD+/四聚体结合。对N-乙酰化的兔肌酶与NAD+结合的荧光滴定表明，赖氨酸-183的乙酰化主要影响前3个宏观的NAD+Kd。第四宏观Kd与天然酶的Kd保持相同的数量级。然而，计算的微观Kd表明，第二、第三和第四NAD+的亲和力是相同的数量级。这一结果表明，赖氨酸-183的乙酰化减敏了辅酶的反协同结合过程。这种化学修饰也影响了这三种酶的砷酸盐激活情况。光谱、超速离心和热稳定性研究没有显示N-乙酰化引起的任何显著构象变化。肌肉或酵母甘油醛-3-磷酸脱氢酶的4个赖氨酸-183残基/四聚体显然同样容易发生N-乙酰化。与酵母酶的反应比与肌肉酶的反应慢得多。赖氨酸-183既不存在于催化部位，也不存在于底物结合部位。然而，这个残基似乎在相邻的两个亚基之间的接触区域中扮演着重要的角色，通过该区域流动与NAD+或砷酸盐结合的构象信息。这一结果与X射线结晶学研究相一致。
The acetylation of the .epsilon.NH2 of lysine-183 of rabbit muscle, sturgeon muscle and yeast glyceraldehyde-3-phosphate dehydrogenases (EC 1.2.1.12) at pH 8.5 by S .fwdarw. N acetyl transfer follows a first-order process. Essentially, 4 lysine residues are modified/enzyme tetramer. When enzymic assays are carried out under saturating conditions of the 3 substrates (NAD+, glyceraldehyde-3-phosphate and arsenate), the N-acetylated rabbit and sturgeon muscle enzymes show values of Vmax which are 90% and 80%, respectively, of that of the native enzymes, while for the N-acetylated yeast enzyme it is only 20%. The Km values of the 3 substrates are not significantly affected by the N-acetylation of the 2 muscle enzymes. However, the N-acetylated yeast enzyme yields an increased Km for NAD+ only. By spectrophotometric titration at the Racker band, it was shown that each of the 3 N-acetylated enzymes still binds 4 NAD+/tetramer. Spectrofluorimetric titration of the binding of NAD+ to the N-acetylated rabbit muscle enzyme revealed that acetylation of lysine-183 affected essentially the 1st 3 macroscopic NAD+ Kd. The 4th macroscopic Kd remained of the same order of magnitude as that found for the native enzyme. However, the calculated microscopic Kd show that the affinities of the 2nd, 3rd and 4th NAD+ are of the same order. This result indicates that acetylation of lysine-183 desensitized the anticooperative binding process of the coenzyme. This chemical modification also affected the profile of arsenate activation for the three enzymes. Spectroscopic, ultracentrifugation and thermostability studies failed to show any significant conformational change due to the N-acetylation. The 4 lysine-183 residues/tetramer of muscle or yeast glyceraldehyde-3-phosphate dehydrogenase are apparently equally accessible to N-acetylation. Reaction with the yeast enzyme is much slower than that with the muscle enzymes. Lysine-183 is implicated neither in the catalytic site nor in the substrate binding site. However, this residue seems to play an important role in the area of contact between the 2 adjacent subunits through which flows conformational information incidental to the binding of NAD+ or arsenate to the protein. This result is in agreement with X-ray crystallographic studies.