Lilley K S, Engel P C
Department of Biochemistry, University of Leicester, England.
Eur J Biochem. 1992 Jul 15;207(2):533-40. doi: 10.1111/j.1432-1033.1992.tb17079.x.
Glutamate dehydrogenase (GDH) of Clostridium symbiosum, like GDH from other species, is inactivated by pyridoxal 5'-phosphate (pyridoxal-P). This inactivation follows a similar pattern to that for beef liver GDH, in which a non-covalent GDH-pyridoxal-P complex reacts slowly to form a covalent complex in which pyridoxal-P is in a Schiff's-base linkage to lysine residues. [formula: see text] The equilibrium constant of this first-order reaction on the enzyme surface determines the final extent of inactivation observed [S. S. Chen and P. C. Engel (1975) Biochem. J. 147, 351-358]. For clostridial GDH, the maximal inactivation obtained was about 70%, reached after 10 min with 7 mM pyridoxal-P at pH 7. In keeping with the model, (a) inactivation became irreversible after reduction with NaBH4. (b) The NaBH4-reduced enzyme showed a new absorption peak at 325 nm. (c) Km values for NAD+ and glutamate were unaltered, although Vmax values were decreased by 70%. Kinetic analysis of the inactivation gave values of 0.81 +/- 0.34 min-1 for k3 and 3.61 +/- 0.95 mM for k2/k1. The linear plot of 1/(1-R) against 1/[pyridoxal-P], where R is the limiting residual activity reached in an inactivation reaction, gave a slightly higher value for k2/k1 of 4.8 +/- 0.47 mM and k4 of 0.16 +/- 0.01 min-1. NADH, NAD+, 2-oxoglutarate, glutarate and succinate separately gave partial protection against inactivation, the biggest effect being that of 40 mM succinate (68% activity compared with 33% in the control). Paired combinations of glutarate or 2-oxoglutarate and NAD+ gave slightly better protection than the separate components, but the most effective combination was 40 mM 2-oxoglutarate with 1 mM NADH (85% activity at equilibrium). 70% inactivated enzyme showed an incorporation of 0.7 mM pyridoxal-P/mol subunit, estimated spectrophotometrically after NaBH4 reduction, in keeping with the 1:1 stoichiometry for the inactivation. In a sample protected with 2-oxoglutarate and NADH, however, incorporation was 0.45 mol/mol, as against 0.15 mol/mol expected (85% active). Tryptic peptides of the enzyme, modified with and without protection, were purified by HPLC. Two major peaks containing phosphopyridoxyllysine were unique to the unprotected enzyme. These peaks yielded three peptide sequences clearly homologous to sequences of other GDH species. In each case, a gap at which no obvious phenylthiohydantoin-amino-acid was detected, matched a conserved lysine position. The gap was taken to indicate phosphopyridoxyllysine which had prevented tryptic cleavage.(ABSTRACT TRUNCATED AT 400 WORDS)
共生梭菌的谷氨酸脱氢酶（GDH）与其他物种的GDH一样，会被磷酸吡哆醛（pyridoxal-P）灭活。这种灭活过程与牛肝GDH的情况类似，即非共价的GDH - 磷酸吡哆醛复合物缓慢反应形成共价复合物，其中磷酸吡哆醛通过席夫碱连接到赖氨酸残基上。[化学式：见原文]酶表面上这个一级反应的平衡常数决定了观察到的最终灭活程度[S. S. 陈和P. C. 恩格尔（1975年），《生物化学杂志》147卷，351 - 358页]。对于梭菌GDH，在pH 7条件下用7 mM磷酸吡哆醛处理10分钟后，最大灭活率约为70%。与该模型一致的是，（a）用硼氢化钠还原后，灭活变得不可逆。（b）硼氢化钠还原后的酶在325 nm处出现一个新的吸收峰。（c）NAD⁺和谷氨酸的Km值未改变，尽管Vmax值降低了70%。对灭活过程的动力学分析得出k3值为0.81±0.34分钟⁻¹，k2/k1值为3.61±0.95 mM。以1/(1 - R)对1/[磷酸吡哆醛]作图（其中R是灭活反应中达到的极限残余活性），得出k2/k1值略高，为4.8±0.47 mM，k4值为0.16±0.01分钟⁻¹。NADH、NAD⁺、2 - 氧代戊二酸、戊二酸和琥珀酸分别对灭活有部分保护作用，最大的作用是40 mM琥珀酸（活性为68%，而对照中为33%）。戊二酸或2 - 氧代戊二酸与NAD⁺的配对组合提供的保护比单独的成分略好，但最有效的组合是40 mM 2 - 氧代戊二酸与1 mM NADH（平衡时活性为85%）。70%灭活的酶在硼氢化钠还原后经分光光度法估计，每摩尔亚基结合了0.7 mM磷酸吡哆醛，这与灭活的1:1化学计量比一致。然而，在用2 - 氧代戊二酸和NADH保护的样品中，结合量为0.45摩尔/摩尔，而预期为0.15摩尔/摩尔（活性为85%）。用和不用保护剂处理的酶的胰蛋白酶肽段通过高效液相色谱法纯化。两个含有磷酸吡哆醛赖氨酸的主要峰是未受保护的酶所特有的。这些峰产生了三个与其他GDH物种序列明显同源的肽序列。在每种情况下，未检测到明显苯硫基乙内酰脲 - 氨基酸的缺口与一个保守的赖氨酸位置匹配。该缺口被认为表明磷酸吡哆醛赖氨酸阻止了胰蛋白酶的切割。（摘要截断于400字）
