McHarg J, Kelly S M, Price N C, Cooper A, Littlechild J A
School of Chemistry, University of Exeter, UK.
Eur J Biochem. 1999 Feb;259(3):939-45. doi: 10.1046/j.1432-1327.1999.00133.x.
Site-specific mutants have been produced in order to investigate the role of proline 204 in the 'hinge' region of yeast phosphoglycerate kinase (PGK). This totally conserved proline has been shown to be the only cis-proline in the high resolution crystal structures of yeast, B. stearothermophilus, T. brucei and T. maritima PGK, and may therefore have a role in the independent folding of the two domains or in the 'hinge' bending of the molecule during catalysis. The residue was replaced by a histidine (Pro204His) and a phenylalanine (Pro204Phe), and the resulting proteins characterised by differential scanning calorimetry (DSC), circular dichroism (CD), tryptophan fluorescence emission and kinetic analysis. Although the secondary and tertiary structure of the Pro204His protein is generally similar to that of the wild-type enzyme as assessed by CD, the enzyme is less stable to heat and guanidinium chloride denaturation than the wild-type. In the denaturation experiments two transitions were observed for both the wild-type and the Pro204His mutant, as have been previously reported for yeast PGK [Missiakas, D., Betton, J.M., Minard, P. & Yon, J.M. (1990) Biochemistry 29, 8683-8689]. The first transition is accompanied by an increase in fluorescence intensity leading to a hyperfluorescent state, followed by the second, corresponding to a decrease in fluorescence intensity. However, for the Pro204His mutant, the first transition proceeded at lower concentrations of guanidinium chloride and the second transition proceeded to the same extent as for the wild-type protein, suggesting that sequence-distant interactions are more rapidly disrupted in this mutant enzyme than in the wild-type enzyme, while sequence-local interactions are disrupted in a similar way. The Michaelis constants (K(m)) for both 3-phospho-D-glycerate and ATP are increased only by three or fourfold, which confirms that, as expected, the substrate binding sites are largely unaffected by the mutation. However, the turnover and efficiency of the Pro204His mutant is severely impaired, indicating that the mechanism of 'hinge' bending is hindered. The Pro204Phe enzyme was shown to be significantly less well folded than the wild-type and Pro204His enzymes, with considerable loss of both secondary and tertiary structure. It is proposed that the proline residue at 204 in the 'hinge' region of PGK plays a role in the stability and catalytic mechanism of the enzyme.
为了研究脯氨酸204在酵母磷酸甘油酸激酶（PGK）“铰链”区域中的作用，已构建了位点特异性突变体。在酵母、嗜热栖热放线菌、布氏锥虫和海栖热袍菌PGK的高分辨率晶体结构中，这个完全保守的脯氨酸是唯一的顺式脯氨酸，因此它可能在两个结构域的独立折叠过程中发挥作用，或者在催化过程中参与分子的“铰链”弯曲。该残基被组氨酸（Pro204His）和苯丙氨酸（Pro204Phe）取代，通过差示扫描量热法（DSC）、圆二色性（CD）、色氨酸荧光发射和动力学分析对所得蛋白质进行了表征。尽管通过CD评估，Pro204His蛋白的二级和三级结构与野生型酶总体相似，但该酶对热和氯化胍变性的稳定性低于野生型。在变性实验中，野生型和Pro204His突变体均观察到两个转变，这与先前报道的酵母PGK情况一致[米西亚卡斯，D.，贝顿，J.M.，米纳德，P. & 扬，J.M.（1990）生物化学29，8683 - 8689]。第一个转变伴随着荧光强度增加，导致超荧光状态，随后是第二个转变，对应于荧光强度降低。然而，对于Pro204His突变体，第一个转变在较低浓度的氯化胍下进行，第二个转变的程度与野生型蛋白相同，这表明与野生型酶相比，该突变酶中序列距离较远的相互作用被破坏得更快，而序列局部的相互作用以类似方式被破坏。3 - 磷酸 - D - 甘油酸和ATP的米氏常数（K(m)）仅增加了三到四倍，这证实了正如预期的那样，底物结合位点在很大程度上不受突变影响。然而，Pro204His突变体的周转和效率严重受损，表明“铰链”弯曲机制受到阻碍。结果表明，Pro204Phe酶的折叠程度明显低于野生型和Pro204His酶，二级和三级结构均有相当程度的丧失。有人提出，PGK“铰链”区域中204位的脯氨酸残基在该酶的稳定性和催化机制中发挥作用。
