Mechanisms of Enzyme Catalysis

本研究将结合位点诱变和结构探针，特别是衰减全反射（ATR） FTIR技术，研究b -内酰胺酶的催化和抑制作用。b内酰胺酶是对青霉素抗生素治疗产生耐药性的主要来源，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目标。尽管进行了大量的研究，但b内酰胺酶的作用机制仍然是一个谜。虽然通过结构、化学和诱变研究已经确定了活性位点残基，并提出了几种可能的机制，但对催化机制尚未达成共识，许多尚未解决的基本问题仍然存在。通过对选定的活性位点突变体的详细动力学和结构表征，我们希望能够回答一些关于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速率分布；保守的Lys73的作用；为什么一些具有野生型酶的底物和一些具有所有底物的突变体表现出分支通路机制（反映在底物诱导的失活或可逆自杀抑制），以及由此产生的失活酰基酶的性质；以及为什么A类B类内酰胺酶的去酰化速率比青霉素靶酶（转肽酶和D Ala、D Ala羧肽酶）快得多的原因。我们期望在这项研究中获得的信息将对了解b内酰胺酶的催化作用机制和设计可能成为潜在抗生素的新抑制剂非常有价值。内酰胺酶是对青霉素抗生素治疗产生耐药性的主要来源，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目标。尽管进行了大量的研究，但b内酰胺酶的作用机制仍然是一个谜。通过对选定的活性位点突变体进行详细的动力学和结构表征，我们希望回答一些关于B内酰胺酶催化机制的具体问题。突变体将使用动力学和结构探针进行表征。我们提出了一种新的方法来获取有关酶-底物复合物（和酶-抑制剂复合物）结构的信息，即使用衰减全反射（ATR） FTIR（傅里叶变换红外光谱），结合固定酶在流动细胞中。通常瞬态中间体可以用低温或使用相对不活跃的突变体来稳定。拟议研究中获得的信息应有助于改进b内酰胺类抗生素的开发。

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.