MAGNETIC RESONANCE STUDIES OF METAL ACTIVATION OF ENZYMIC REACTIONS OF NUCLEOTIDES AND OTHER PHOSPHATE SUBSTRATES.
MAGNETIC RESONANCE STUDIES OF METAL ACTIVATION OF ENZYMIC REACTIONS OF NUCLEOTIDES AND OTHER PHOSPHATE SUBSTRATES.
复制标题
核苷酸和其他磷酸盐底物酶反应金属活化的磁共振研究。
DOI：
10.1021/bi00904a001
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发表时间：
1963
期刊：
影响因子：
2.9
通讯作者：
M. Cohn
中科院分区：
文献类型：
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作者：
M. Cohn
核苷三磷酸的所有反应都需要二价金属离子，利用锰离子的顺磁性研究了金属、底物和酶的相互作用。顺磁性离子Mn++对反应混合物中水质子的核磁共振弛豫速率的影响对所形成的金属络合物的类型非常敏感。在与蛋白质形成二元或三元复合物时，测量到相对于锰的水溶液（在1 × 10-4 m处容易观察到）的效果增强。用这种技术研究了肌酸激酶、3-磷酸甘油酸激酶、己糖激酶和丙酮酸激酶等四种晶体激酶与其各自底物的相互作用，以及另一种金属需要酶酵母烯醇化酶与底物的相互作用。这些酶分为两组：（1）二元复合物（Mn-酶）的增强»三元复合物（Mn-酶-底物）的增强，和（2）三元复合物的增强»二元复合物。第一组包括烯醇化酶和丙酮酸激酶，第二组包括肌酸激酶和3-磷酸甘油酸激酶。与酵母己糖激酶，需要酶-葡萄糖-Mn-ADP的四元复合物来引起增强。对于肌酸激酶，增强数据表明，锰并不作为酶和底物之间的桥梁，而是仅与核苷酸络合，然后金属核苷酸与酶结合。三元配合物的电子自旋共振谱证实了这种构型。增强测量已被用于定量测定金属酶，金属底物，和底物金属酶的缔合常数。对于那些酶，如烯醇化酶和丙酮酸激酶，其中的证据表明，金属离子结合在活性位点，由于锰酶的水的质子弛豫速率的增强是一个特定的网站的特点，不仅可以用于滴定的网站，但也是一个非常敏感的指标，在构型的变化只发生在活性位点。
The requirement of a divalent metal ion is common to all reactions of nucleoside triphosphates, and advantage has been taken of the paramagneticproperty of the manganous ion to study interactions of metal, substrate, andenzyme. The effect of the paramagnetic ion Mn++ on the relaxation rate of the nuclear magnetic resonance of water protons in the reaction mixture is very sensitive to the type of metal complex formed. Upon formation of binary or ternary complexes with proteins, enhancement of the effect relative to the manganese aquocation (easily observable at 1 X 10-4 m) hasbeen measured. The interactions of four crystalline kinases, creatine kinase, 3-phosphoglycerate kinase, hexokinase, and pyruvate kinase, with their respec-tive substrates and’+ have been investigated with this technique as well as another metalrequiring enzyme, yeast enolase. These enzymes fall into two groups:(1) enhancement of the binary complex (Mn-enzyme)» enhancement of ternary complex (Mn-enzyme-substrate), and (2) enhancement of ternary complex» binary complex. The first group includes enolase and pyruvate kinase, and the second group includes creatine kinase and 3-phosphoglycerate kinase. With yeast hexokinase a quaternary complex of enzyme-glucose-Mn-ADP was re-quired to cause enhancement. For creatine kinase the enhancement data suggested that man-ganese does notact as a bridge betweenenzyme and substrate, but is complexed only to the nucleotide, and the metal-nucleotide is then bound to enzyme. This configuration of the ternary complex was verified by electron-spin-resonance spectra of the binary and ternary com-plexes. The measurement of enhancement has been used for quantitative determination of association constants for metal enzyme, metal substrate, and substrate-metal enzyme. For those enzymes such as enolase and pyruvate kinase, where the evidence indicatesthat the metal ion is bound at the active site, the enhancement of theproton relaxation rate of water due to the Mn-enzyme is characteristic of a particular site and not only can be used for titration of the site but is also a very sensitive indicator of changes in configuration occurring solelyat the active site.