XAFS STUDIES OF INTERMEDIATES IN ZINC PROTEASE CATALYSIS

锌蛋白酶催化中间体的 XAFS 研究

• 批准号: 3307057
• 负责人: DAVID S AULD
• 金额: $ 22.85万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3307057
• 关键词: X ray spectrometry; active sites; carboxypeptidase; chemical kinetics; cold temperature; enzyme activity; enzyme structure; enzyme substrate complex; high performance liquid chromatography; metal complex; zinc

Direct verification of enzyme mechanisms requires knowledge of the structure of intermediates in catalysis. Metalloenzymes like carboxypeptidase A provide a unique opportunity in this regard because the metal is involved in catalysis and the structure of the metal binding site can be investigated in solution by X-ray Absorption Fine Structure, XAFS analysis. Experimentally, concurrent measurements of both structural and kinetic characteristics of such intermediates requires a lengthening of their very short lifetimes, e.g. by reducing their rates of formation and breakdown. This can be achieved by carrying out the reaction at low temperature and using rapid mixing devices. The ideal system is composed of several features all of which have the same fundamental objective i.e. detection and characterization of reaction intermediates without alteration of the reaction pathway. Of particular importance to these studies are the 1) means to detect structural changes, 2) ability to rapidly assay the enzyme at subzero temperatures and 3) crysolvent employed. Each of these features must be considered carefully for the enzyme under consideration. For CPD A three prominent features are its requirement for zinc for activity, its high reactivity toward N-terminal blocked peptides and depsipeptides and its solubility in salt solutions. Both of the former features can serve as a basis for creating suitable chromophoric probes. Substituting Co for Zn places a chromophoric atom at the active site of the enzyme which is sensitive to structural changes and the use of a fluorescent dansyl substrate blocking group can serve as the basis of a radiatioriless energy transfer assay of enzyme activity. These probes can aid in identifying substrates whose intermediates can be stabilized either by freeze quenching or equilibrium trapping at subzero temperatures, so that their structure can be determined by XAFS. In parallel experiments HPLC analysis will determine if the scissle bond is broken in the freeze quenched intermediate. XAFS allows structural comparisons of the metal binding site complexed with substrates or inhibitors for the solution and crystalline forms of both ZnCPD and CoCPD. The structure of the intermediates of CPD A will be compared to those obtained for other zinc proteases which have different zinc binding sites. The structure of these active site metal coordination complexes should be useful in assigning electronic transitions that occur in Co visible absorption, CD and MCD spectra to changes in the geometry of the complex as a whole or perhaps to movement of a single ligand.

直接验证酶的机制需要知识