CRYSTALLOGRAPHIC STRUCTURE OF A PHOSPHONATE DERIVATIVE OF THE ENTEROBACTER-CLOACAE P99 CEPHALOSPORINASE - MECHANISTIC INTERPRETATION OF A BETA-LACTAMASE TRANSITION-STATE ANALOG
CRYSTALLOGRAPHIC STRUCTURE OF A PHOSPHONATE DERIVATIVE OF THE ENTEROBACTER-CLOACAE P99 CEPHALOSPORINASE - MECHANISTIC INTERPRETATION OF A BETA-LACTAMASE TRANSITION-STATE ANALOG
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DOI：
10.1021/bi00188a004
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发表时间：
1994-06-07
期刊：
影响因子：
2.9
通讯作者：
KNOX, JR
中科院分区：
文献类型：
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作者：
LOBKOVSKY, E;BILLINGS, EM;KNOX, JR
在2.3埃的分辨率下，获得了阴沟肠杆菌P99头孢菌素酶（β -内酰胺酶）与膦酸酯单酯抑制剂m-羧基苯基[[N-[（对碘苯基）乙酰基]氨基]甲基]膦酸盐反应形成的配合物的晶体结构。结构表明该抑制剂磷酸化了活性位点丝氨酸（Ser64），失去了间羧基酚离去基。抑制剂以一种可以用过渡态类似物解释的方式定位于活性位点。芳基乙酰氨基侧链的位置与青霉素识别酶的类似物β -内酰胺基络合物的位置一致，酰胺基氢键与Ser318 （B3 β链）的主链羰基以及Gln120和Asn152的酰胺相连。该侧链与蛋白质的氢键不对称以及苯基的2倍无序性支持C类β -内酰胺酶在底物特异性方面表现出相当大的广度。一个磷酰氧原子在氧阴离子空穴中，与Ser318和Ser64的主链NH基团成氢键，而另一个氧是溶剂化的，不在任何氨基酸侧链的氢键距离内。离溶剂化氧原子最近的活性位官能团是Tyr150羟基（3.4埃）；Lys67和Lys315距离相当远（分别为4.3和5.7埃）。相反，Tyr150和Lys67与Ser640 γ（2.9和3.3埃）更密切相关。这种排列被解释为在催化的去酰化步骤中四面体中间体分解的过渡态，其中Tyr150酚似乎是最可能的一般酸。因此，Oefner等人提出，Tyr150作为苯氧阴离子，将是酰化反应的一般碱催化剂[Nature(1990) 343,284 -288]。该结构与a类β -内酰胺酶的类似膦酸衍生物的结构进行了比较[Chen et al. (1993) J. Mol. Biol. 234,165 -178]，并进行了机制比较。与丝氨酸蛋白酶相反，丝氨酸-内酰胺酶对膦酸盐单阴离子抑制的敏感性得到静电计算的支持，静电计算显示，只有在-内酰胺酶的催化位点才有净正电位。
The crystal structure of a complex formed on reaction of the Enterobacter cloacae P99 cephalosporinase (beta-lactamase) with a phosphonate monoester inhibitor, m-carboxyphenyl [[N-[(p-iodophenyl)acetyl]amino]methyl]phosphonate, has been obtained at 2.3-Angstrom resolution. The structure shows that the inhibitor has phosphonylated the active site serine (Ser64) with loss of the m-carboxyphenol leaving group. The inhibitor is positioned in the active site in a way that can be interpreted in terms of a transition-state analog. The arylacetamido side chain is placed as anticipated from analogues beta-lactamoyl complexes of penicillin-recognizing enzymes, with the amido group hydrogen-bonded to the backbone carbonyl of Ser318 (of the B3 beta-strand) and to the amides of Gln120 and Asn152. There is support in the asymmetry of the hydrogen bonding of this side chain to the protein and in the 2-fold disorder of the benzyl group for the considerable breadth in substrate specificity exhibited by class C beta-lactamases. One phosphonyl oxygen atom is in the oxyanion hole, hydrogen-bonded to main-chain NH groups of Ser318 and Ser64, while the other oxygen is solvated, not within hydrogen-bonding distance of any amino acid side chain. The closest active site functional group to the solvated oxygen atom is the Tyr150 hydroxyl group (3.4 Angstrom); Lys67 and Lys315 are quite distant (4.3 and 5.7 Angstrom, respectively). Rather Tyr150 and Lys67 are more closely associated with Ser640 gamma (2.9 and 3.3 Angstrom). This arrangement is interpreted in terms of the transition state for breakdown of the tetrahedral intermediate in the deacylation step of catalysis, where the Tyr150 phenol seems the most likely general acid. Thus, Tyr150, as the phenoxide anion, would be the general base catalyst in acylation, as proposed by Oefner et al. [Nature (1990) 343, 284-288]. The structure is compared with that of a similar phosphonate derivative of a class A beta-lactamase [Chen et al. (1993) J. Mol. Biol. 234, 165-178], and mechanistic comparisons are made. The sensitivity of serine beta-lactamases, as opposed to serine proteinases, toward inhibition by phosphonate monoanions is supported by electrostatic calculations showing a net positive potential only in the catalytic sites of the beta-lactamases.