The high-resolution structures of the neutral and the low pH crystals of aminopeptidase from Aeromonas proteolytica.
Summary (3 min read)
- Bridged bimetallic enzymes contain two metal ions held in close proximity by a protein ligand and/or an oxygen atom, usually from bulk solvent, that spans two metal ions.
- It has been proposed that both metal ions are necessary to recognize and bind substrate, to activate the attacking nucleophile, and to stabilize intermediates of the reaction.
- A chemical reaction mechanism has been proposed for AAP  in which the substrate binds to AAP by first coordinating the carbonyl oxygen of the N-terminal amino acid to Zn1 followed by the coordination of the N-terminal amine to Zn2.
- In the absence of electron density corresponding to hydrogen atoms, precise coordination distances can be used to establish the identity of the bridging water species by comparing its Zn-O bond distances.
- All chemicals used in this study were purchased commercially and were of the highest quality available.
- AAP was purified from a stock culture kindly provided by Céline Schalk.
- Cultures were grown according to the previously reported protocol with minor modifications  to the growth media.
- Purified enzyme was stored at −196 °C until needed.
- The protein was crystallized using the conditions reported previously  with the exception that Tris was replaced with HEPES  .
Data collection and processing
- An AAP crystal was removed from the hanging drop, soaked in mother liquor containing 10% glycerol for 1 min, coated with Paratone-N oil, mounted in a 0.5-mm Hampton Research CryoLoop, and flash-cooled to −173 °C in liquid nitrogen.
- Second, the low-resolution data were collected by reducing the exposure time to 0.5 s, moving the 2θ angle to 0°, and increasing the sample-to-detector distance to 200 mm.
- Refinement for both data sets was carried out using the software package CNS  followed by SHELX.
- The structure was refined almost to convergence.
- The blocks contained overlapping residues so that every ESD could be estimated with all contributing atoms being refined in at least one of the refinement cycles.
Results and discussion
- The reaction mechanism step at which deprotonation of the bridging solvent molecule occurs, the most likely candidate to function as the nucleophile, is unknown.
- Of the 184 peaks associated with carbon atoms, a modest increase in the number of potential C α hydrogen peaks was observed when increasing the resolution from 1.20 to 0.95 Å, while the number of peaks associated with side chain carbons and amide nitrogen atoms doubled (Table 2 ).
- The normal motion of the heavier atoms that associate with hydrogen atoms can reduce the hydrogen contribution to background noise, making it impossible to observe them in a Fourier difference map and difficult to reproduce them from one data set to the next.
- The calculated estimated standard deviation associated with each distance is shown in parentheses Electron density corresponding to potential hydrogen atoms in the active site was not observed in the Fourier difference map with coefficients F obs−F calc; however, the protonation state of the bridging oxygen species can be determined by comparing Zn-O distance standards found in the CSD  and those studied by computational methods  with the precise Zn-O distances obtained in this structure.
- If the bridging oxygen species is OH − and Glu151 is the proton acceptor  , then the dangling oxygen of Glu152 may serve to stabilize the bridging hydroxide ion or the protonated carboxylate side chain by forming a hydrogen-bonding interaction with them.
Fig. 2. Schematic of the active site of native aminopeptidase from Aeromonas proteolytica (AAP)
- For Asp179, there is a 0.04-Å difference in bond length between the two carbon-oxygen bonds of the side chain carboxylate, with the distance to the inner-sphere oxygen being slightly shorter than the dangling oxygen distance.
- The carbon-oxygen distance to the inner-sphere oxygen of Asp179 is significantly shorter than the carbon-oxygen distances for the metal ligated oxygen atoms of Glu152 and Asp117 (Δd=0.05-0.07 Å) and is consistent with that for C=O distances measured in small molecules, implying that this bond has more double-bond character.
- If the bridging oxygen in the native enzyme is an OH − , as identified earlier herein, then the level of precision obtained at high resolution should make it possible to observe the 0.1-0.2-Å change that should occur in Zn-O distances when the bridging oxygen changes its protonation state from OH − to OH2.
- A coordination number change is the most likely cause since the Zn2-O distance remains consistent with that observed for Zn-OH, suggesting that the bridging solvent species has not taken on a second proton.
- A result of the increase in the Zn1-OH bond distance is the strong Zn2-OH interaction (1.93 Å).
Movement of metal ions during catalysis
- During the course of the enzymatic reaction, the distances of the metal ions to their ligands may change in accordance with the chemical state of the substrate/intermediate.
- At 1.8-Å resolution, the resolution at which several crystal structures of AAP have been studied, it is impossible to assess the thermal motion of the atoms because of the isotropic treatment of the thermal parameters.
- The anisotropic treatment of thermal motion has indicated that the active site is actually asymmetric with one metal binding site being more rigid than the other.
- This relative increase in the thermal motion of the Zn1 nonbridging ligands can be attributed to the lack of possible protein hydrogenbonding partners for Glu152 and His256.
- LPA coordinates to both zinc ions and pushes Zn1 0.60 Å away from its position in the native enzyme, while Zn2 remains close to its original position.
Insight into the chemical mechanism
- The structures of AAP presented herein have provided additional evidence for the proposed reaction mechanism of AAP that was not obtainable from lower-resolution X-ray structures.
- Glu151, the proton acceptor in this activation step  , donates a proton to the penultimate nitrogen of the product later in the reaction cycle.
- On the basis of kinetics, spectroscopic and X-ray crystallographic data [6, 21] it has been proposed that substrate coordinates to the bimetallic center in a stepwise fashion with the carbonyl oxygen of the N-terminal amino acid first coordinating to Zn1 followed by the coordination of the free amine to Zn2 (Fig. 5 , species 2 and 3).
- Spectroscopic evidence for Co(II)-substituted AAP indicates that the two metal ions in the AAP active site bind in a sequential fashion and that upon introduction of substrate the coordination geometry of the first metal binding site changes from four to five coordinate.
- In addition, the bridging solvent molecule becomes terminal and is bound to the first metal binding site.
Fig. 5. Proposed chemical reaction mechanisms for AAP
- On the basis of X-ray crystallographic and spectroscopic studies of AAP complexed with the substrate analog inhibitor 1-butaneboronic acid (BuBA) , the metal ion thought to play the catalytic role was identified as Zn1.
- A molecule of water was also observed near the active site.
- It may provide information about the differences in the behavior of the two metal ions in the nearly symmetrical AAP active site.
- For intermediate 3a to form, the second substrate binding step would involve coordination of the N-terminal amine to Zn2 followed by breaking of the Zn2-OH bond, consistent with spectroscopic and X-ray crystallographic data.
- Distinguishing the contributions of the two identical Zn(II) ions in AAP is an important question since AAP displays 80% of its total activity level with only a single metal ion bound.
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Cites background or methods or result from "The high-resolution structures of t..."
...A similar pattern of Glu-carbonyl interactions is observed in structures of the above-mentioned aminopeptidases (24,47-49) (Figure 6)....
...The authors carried out DFT calculations (using B3LYP functional) on the model of the active site of AAP (so-called cluster model), which was derived from an existing crystal structure of a substrate-free AAP (24) with a model dipeptide (Ala-Ala) docked as a substrate....
...Despite the above-mentioned efforts, the reaction mechanism of GCPII is not fully understood in detail, although some information can be derived from extrapolation of the known reaction mechanism of the related AAP, for which more experimental (X-ray crystallography, kinetics, spectroscopy, and site-directedmutagenesis) and computational data are available (24,25)....
...Our experimental and theoretical data on the GCPII reaction mechanism are consistent with findings of others on related aminopeptidases (2,24-26) and suggest a common mechanism of substrate hydrolysis by this class of enzymes....
...Similar distance variability of Znhas beennotedduring the reaction cycle ofAAP, and it has been suggested that positional flexibility of metal ions is required as a response to a constantly changing environment in the active site of binuclear enzymes (24)....
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