Copik Alicja J, Nocek Boguslaw P, Swierczek Sabina I, Ruebush Shane, Jang Se Bok, Meng Lu, D'souza Ventris M, Peters John W, Bennett Brian, Holz Richard C
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300, USA.
Biochemistry. 2005 Jan 11;44(1):121-9. doi: 10.1021/bi048123+.
Methionine aminopeptidases (MetAPs) are ubiquitous metallohydrolases that remove the N-terminal methionine from nascent polypeptide chains. Although various crystal structures of MetAP in the presence of inhibitors have been solved, the structural aspects of the product-bound step has received little attention. Both perpendicular- and parallel-mode electron paramagnetic resonance (EPR) spectra were recorded for the Mn(II)-loaded forms of the type-I (Escherichia coli) and type-II (Pyrococcus furiosus) MetAPs in the presence of the reaction product l-methionine (L-Met). In general, similar EPR features were observed for both [MnMn(EcMetAP-I)]-L-Met and [MnMn(PfMetAP-II)]-L-Met. The observed perpendicular-mode EPR spectra consisted of a six-line hyperfine pattern at g = 2.03 (A = 8.8 mT) with less intense signals with eleven-line splitting at g = 2.4 and 1.7 (A = 4.4 mT). The former feature results from mononuclear, magnetically isolated Mn(II) ions and this signal are 3-fold more intense in the [MnMn(PfMetAP-II)]-L-Met EPR spectrum than in the [MnMn(EcMetAP-I)]-L-Met spectrum. Inspection of the EPR spectra of both [MnMn(EcMetAP-I)]-L-Met and [MnMn(PfMetAP-II)]-L-Met at 40 K in the parallel mode reveals that the [Mn(EcMetAP-I)]-L-Met spectrum exhibits a well-resolved hyperfine split pattern at g = 7.6 with a hyperfine splitting constant of A = 4.4 mT. These data suggest the presence of a magnetically coupled dinuclear Mn(II) center. On the other hand, a similar feature was not observed for the [MnMn(PfMetAP-II)]-L-Met complex. Therefore, the EPR data suggest that L-Met binds to [MnMn(EcMetAP-I)] differently than [MnMn(PfMetAP-II)]. To confirm these data, the X-ray crystal structure of [MnMn(PfMetAP-II)]-L-Met was solved to 2.3 A resolution. Both Mn1 and Mn2 reside in a distorted trigonal bipyramidal geometry, but the bridging water molecule, observed in the [CoCo(PfMetAP-II)] structure, is absent. Therefore, L-Met binding displaces this water molecule, but the carboxylate oxygen atom of L-Met does not bridge between the two Mn(II) ions. Instead, a single carboxylate oxygen atom of L-Met interacts with only Mn1, while the N-terminal amine nitrogen atom binds to M2. This L-Met binding mode is different from that observed for L-Met binding [CoCo(EcMetAP-I)]. Therefore, the catalytic mechanisms of type-I MetAPs may differ somewhat from type-II enzymes when a dinuclear metalloactive site is present.
甲硫氨酸氨肽酶（MetAPs）是普遍存在的金属水解酶，可从新生多肽链中去除N端甲硫氨酸。尽管已解析了MetAP在存在抑制剂情况下的各种晶体结构，但产物结合步骤的结构方面却很少受到关注。在反应产物L-甲硫氨酸（L-Met）存在的情况下，记录了I型（大肠杆菌）和II型（嗜热栖热菌）MetAP的锰（II）负载形式的垂直和平行模式电子顺磁共振（EPR）光谱。一般来说，[MnMn（EcMetAP-I）]-L-Met和[MnMn（PfMetAP-II）]-L-Met都观察到了相似的EPR特征。观察到的垂直模式EPR光谱在g = 2.03（A = 8.8 mT）处由六线超精细模式组成，在g = 2.4和1.7（A = 4.4 mT）处有强度较低的信号，伴有十一线分裂。前一个特征来自单核、磁隔离的锰（II）离子，并且该信号在[MnMn（PfMetAP-II）]-L-Met EPR光谱中比在[MnMn（EcMetAP-I）]-L-Met光谱中强3倍。在40 K下以平行模式检查[MnMn（EcMetAP-I）]-L-Met和[MnMn（PfMetAP-II）]-L-Met的EPR光谱发现，[Mn（EcMetAP-I）]-L-Met光谱在g = 7.6处呈现出分辨率良好的超精细分裂模式，超精细分裂常数A = 4.4 mT。这些数据表明存在磁耦合双核锰（II）中心。另一方面，[MnMn（PfMetAP-II）]-L-Met配合物未观察到类似特征。因此，EPR数据表明L-Met与[MnMn（EcMetAP-I）]的结合方式与[MnMn（PfMetAP-II）]不同。为了证实这些数据，将[MnMn（PfMetAP-II）]-L-Met的X射线晶体结构解析到2.3 Å分辨率。Mn1和Mn2都处于扭曲的三角双锥几何构型，但在[CoCo（PfMetAP-II）]结构中观察到的桥连水分子不存在。因此，L-Met的结合取代了这个水分子，但L-Met的羧酸根氧原子并未在两个锰（II）离子之间桥连。相反，L-Met的单个羧酸根氧原子仅与Mn1相互作用，而N端胺氮原子与M2结合。这种L-Met的结合模式与观察到的L-Met与[CoCo（EcMetAP-I）]的结合模式不同。因此，当存在双核金属活性位点时，I型MetAPs的催化机制可能与II型酶略有不同。
