Mechanisms of Enzyme Catalysis

酶催化机制

• 批准号: 9418724
• 负责人: Anthony Fink
• 金额: $ 27.4万
• 依托单位: University of California-Santa Cruz
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9418724&HistoricalAwards=false
• 关键词: Mechanisms; Enzyme; Catalysis

The proposed research involves studies on b lactamase catalysis and inhibition, using a combination of site directed mutagenesis and structural probes, especially attenuated total reflectance (ATR) FTIR. b Lactamases are the major source of resistance to penicillin antibiotic therapy, an increasingly common health problem, and are thus potential targets for the design of new antibiotics. In spite of considerable study the mechanism of action of the b lactamases remains somewhat of an enigma. Although the active site residues have been identified through structural, chemical and mutagenesis studies, and several possible mechanisms have been proposed, there is no consensus on the catalytic mechanism, and many outstanding fundamental questions remain. Through detailed kinetic and structural characterization of selected active site mutants we hope to answer a number of specific questions regarding the b lactamase catalytic mechanism, including which ionizing groups are responsible for the bell shaped pH rate profiles; the role of the conserved Lys73; why some substrates with wild type enzy me, and some mutants with all substrates, show a branched path mechanism (reflected in substrate induced inactivation or reversible suicide inhibition), and the nature of the resulting inactive acyl enzyme species; and the reason why the deacylation rate is so much faster in the Class A B lactamases compared to the penicillin target enzymes (transpept-idases and D Ala,D Ala carboxypeptidases). We anticipate that the inform-ation learned in this investigation will be very valuable in understanding how b lactamase catalysis works and in the design of new inhibitors which would be potential antibiotics. %%% Lactamases are the major source of resistance to penicillin antibiotic therapy, an increa ingly common health problem, and are thus potential targets for the design of new antibiotics. In spite of considerable study, the mechanism of action of the b lactamases remains somewhat of an enigma. Through detailed kinetic and strutural characterization of selected active site mutants we hope to answer a number of specific questions regarding the B lactamase catalytic mechanism. The mutants will be characterized using both kinetic and stnuctural probes. We propose a novel approach to obtaining information about the structure of enzyme-substrate complexes (and enzyme-inhibitor complexes), namely the use of attenuated total reflectance (ATR) FTIR (Fourier Transform Infrared spectroscopy),in conjunction with immobilized enzyme in a flow cell. Normally transient intermediates will be stabilized either with low temperatures, or by using relatively inactive mutants. The information obtained in the proposed studies should facilitate the development of improved b lactam antibiotics.

本研究拟采用定点突变和结构探针相结合的方法，特别是衰减全反射（ATR）FTIR，对B内酰胺酶的催化和抑制作用进行研究。 B内酰胺酶是对青霉素抗生素治疗耐药性的主要来源，这是一个日益普遍的健康问题，因此是设计新抗生素的潜在靶点。尽管进行了大量的研究，但B内酰胺酶的作用机制仍是一个谜。虽然活性位点残基已被确定通过结构，化学和诱变研究，并提出了几种可能的机制，有没有共识的催化机制，许多悬而未决的基本问题仍然存在。通过对所选活性位点突变体的详细动力学和结构表征，我们希望回答一些关于B内酰胺酶催化机制的具体问题，包括哪些电离基团负责钟形pH速率曲线;保守的Lys 73的作用;为什么有些底物具有野生型酶，而有些突变体具有所有底物，（反映在底物诱导的失活或可逆的自杀抑制中），以及所得的失活酰基酶种类的性质;以及A类B内酰胺酶的脱酰速率比青霉素靶酶快得多的原因（转肽酶和D Ala，D Ala羧肽酶）。我们期望本研究所获得的信息对于理解B内酰胺酶的催化作用和设计具有潜在抗生素潜力的新型抑制剂具有重要价值。 %%% 内酰胺酶是青霉素类抗生素耐药性的主要来源，是一种日益普遍的健康问题，因此是设计新抗生素的潜在靶点。尽管进行了大量的研究，但B内酰胺酶的作用机制仍是一个谜。通过对所选活性位点突变体的详细动力学和结构表征，我们希望回答一些关于B内酰胺酶催化机制的具体问题。将使用动力学和结构探针表征突变体。我们提出了一种新的方法来获得有关酶-底物复合物（和酶-抑制剂复合物）的结构的信息，即使用衰减全反射（ATR）FTIR（傅里叶变换红外光谱），结合固定化酶在流动池中。通常短暂的中间体将通过低温或通过使用相对无活性的突变体来稳定。在拟议的研究中获得的信息应有助于改进B内酰胺抗生素的开发。