ALTERING ENZYMATIC-ACTIVITY - RECRUITMENT OF CARBOXYPEPTIDASE ACTIVITY INTO AN RTEM BETA-LACTAMASE PENICILLIN-BINDING PROTEIN-5 CHIMERA
ALTERING ENZYMATIC-ACTIVITY - RECRUITMENT OF CARBOXYPEPTIDASE ACTIVITY INTO AN RTEM BETA-LACTAMASE PENICILLIN-BINDING PROTEIN-5 CHIMERA
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DOI：
10.1073/pnas.87.7.2823
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发表时间：
1990-04-01
影响因子：
11.1
通讯作者：
RICHARDS, JH
中科院分区：
文献类型：
--
作者：
CHANG, YH;LABGOLD, MR;RICHARDS, JH
D-Ala-D-Ala羧肽酶/转肽酶（青霉素结合蛋白，PBP）与A β类有相当大的结构同源性-内酰胺酶（EC 3.5.2.6），尽管这些. β. -内酰胺酶没有可观察到的D-Ala-D-Ala羧肽酶活性。目的是招募这种活动进入β-在RTEM-1 β-内酰胺酶的背景下，我们通过插入大肠杆菌PBP-5的28个氨基酸片段代替RTEM-1 β-内酰胺酶的相应区域制备了嵌合蛋白。内酰胺酶 如此插入的片段包含两个家族中保守的残基：Ser-70，其在β-半乳糖苷酶过程中形成酰基酶中间体。内酰胺水解和Lys-73，其存在已被证明是催化所必需的。这种嵌合体涉及18个残基的变化，并产生与亲本残基相差7%的蛋白质。而RTEM β-内酰胺酶没有D-Ala-D-Ala羧肽酶活性，嵌合体的活性是显著的，并且事实上是PBP-5对二乙酰基-L-Lys-D-Ala-D-Ala的活性的约1%;就活化自由能而言，嵌合体将反应的过渡态稳定在PBP-5所实现的稳定的约2.7 kcal/mol内。此外，该嵌合体仅在羧基末端酰胺键处催化水解，该羧基末端酰胺键是被D-Ala-D-Ala羧肽酶切割的位点。尽管含有所有那些在整个A β类中保守的残基，内酰胺酶，并且被认为是β-内酰胺酶所必需的。内酰胺酶活性，嵌合体具有显著降低的活性（约10-5）对青霉烷类如青霉素和氨苄青霉素作为底物。作为催化剂，该嵌合体显示出约400 μ g的诱导期。30分钟，反映为从无活性前体到活性酶的低构象重排。
The D-Ala-D-Ala carboxypeptidases/transpeptidase (penicillin-binding proteins, PBPs) share considerable structural homology with class A .beta.-lactamases (EC 3.5.2.6), although these .beta.-lactamases have no observable D-Ala-D-Ala carboxypeptidase activity. With the objective of recruiting such activity into a .beta.-lactamase background, we have prepared a chimeric protein by inserting a 28-amino acid segment of PBP-5 of Escherichia coli in place of the corresponding region of the RTEM-1 .beta.-lactamase. The segment thus inserted encompasses two residues conserved in both families: Ser-70, which forms the acyl-enzyme intermediate during .beta.-lactam hydrolysis, and Lys-73, whose presence has been shown to be necessary for catalysis. This chimera involves changes of 18 residues and gives a protein that differs at 7% of on the residues from the parent. Whereas RTEM .beta.-lactamase has no D-Ala-D-Ala carboxypeptidase activity, that of the chimera is significant and is, in fact, about 1% the activity of PBP-5 on diacetyl-L-Lys-D-Ala-D-Ala; in terms of free energy of activation, the chimera stabilizes the transition state for the reaction to within about 2.7 kcal/mol of the stabilization achieved by PBP-5. Furthermore, the chimera catalyzes hydrolysis exlusively at the carboxyl-terminal amide bond which is the site of cleavage by D-Ala-D-Ala carboxypeptidase. Though containing all those residues that are conserved throughout class A .beta.-lactamases and are thought to be essential for .beta.-lactamase activity, the chimera has considerably reduced activity (.apprxeq. 10-5) on penams such as penicillins and ampicillins as substrates. As a catalyst, the chimera shows an induction period of .apprxeq. 30 min, reflecting as low conformational rearrangement from an inactive precursor to the active enzyme.