Structural Mechanics of the pH-dependent Activity of beta-Carbonic Anhydrase from Mycobacterium tuberculosis (original) (raw)
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Journal of Molecular Biology, 2008
Mycobacterium tuberculosis ornithine carbamoyltransferase (Mtb OTC) catalyzes the sixth step in arginine biosynthesis; it produces citrulline from carbamoyl phosphate (CP) and ornithine (ORN). Here, we report the crystal structures of Mtb OTC in orthorhombic (form I) and hexagonal (form II) space groups. The molecules in form II are complexed with CP and L-norvaline (NVA); the latter is a competitive inhibitor of OTC. The asymmetric unit in form I contains a pseudo hexamer with 32 point group symmetry. The CP and NVA in form II induce a remarkable conformational change in the 80s and the 240s loops with the displacement of these loops towards the active site. The displacement of these loops is strikingly different from that seen in other OTC structures. In addition, the ligands induce a domain closure of 4.4°in form II. Sequence comparison of activesite residues of Mtb OTC with several other OTCs of known structure reveals that they are virtually identical. The interactions involving the active-site residues of Mtb OTC with CP and NVA and a modeling study of ORN in the form II structure strongly rule out an earlier proposed mechanistic role of Cys264 in catalysis and suggest a possible mechanism for OTC. Our results strongly support the view that ORN with an already deprotonated N ε atom is the species that binds to the enzyme and that one of the phosphate oxygen atoms of CP is likely to be involved in accepting a proton from the doubly protonated N ε atom of ORN. We have interpreted this deprotonation as part of the collapse of the transition state of the reaction.
The enzymatic mechanism of carboxypeptidase: A molecular dynamics study
Proteins: Structure, Function, and Genetics, 1994
An MD simulation of the system carboxypeptidase A (CPA) with the tetrapeptide Val-Leu-Phe-Phe has been performed in order to learn about the substrate disposition just prior to nucleophilic attack. We have explored the model in which the substrate does not substitute the zinc-coordinated water (the "water" mechanism). The simulations do suggest as feasible that the Zn-OH, group performs a nucleophilic attack on the Phe-Phe peptidic bond. We have also investigated the model in which the carbonyl oxygen displaces the zinc-coordinated water. In this case the substrate and Glu-270 orient themselves to allow an anhydride intermediate during the peptidic bond cleavage (the "anhydride" mechanism). Based on the results of the simulations, both "water" and "anhydride" mechanisms are structurally feasible, although the former model seems more probable on chemical grounds. o 1984 Wiley-Liss, Inc.
Active-Site Mechanisms of the Carbonic Anhydrases
Annals of The New York Academy of Sciences, 1984
During the last twenty years, carbonic anhydrase (CA) (EC 4.2.1.1) has played a significant role in the continuing illumination of the principles underlying enzyme activity. In addition to its intrinsic physiological importance, CA is an extremely convenient enzyme for study. The isolated zinc metalloenzyme is stable in solution and during storage; in fact, the highly active form, CA 11, can tolerate pH values in the range 5.5 to 12. As a result of its pronounced catalytic power and robust constitution, CA was transformed into a veritable "laboratory" in which enzyme catalysis was rigorously tested and explored employing the numerous principles of solution chemistry.
Dynamical structure of carboxypeptidase A
Journal of Molecular Biology, 1989
Structural fluctuations of the apoenzyme form of carboxypeptidase A (EC 3.4.12.2) have been evaluated on the basis of molecular dynamics. The Konnert-Hendrickson refined coordinates of 2437 non-hydrogen atoms of the 307 amino acid residues derived from the X-ray structure of the holoenzyme served as the molecular model together with 548 calculated polar hydrogen atoms and 25 buried solvent molecules. Molecular dynamics simulations were carried out at 277 K, and the averaged structural properties of the protein were evaluated for the terminal 20 picosecond portion of a 48 picosecond trajectory. The average atomic displacement from the initial X-ray structure was 2.49 A for all atoms and 1.79 A for C" atoms. The average root-mean-square (r.m.s.) fluctuation of all atoms was 0.67 A as compared to 0.54 A evaluated from the X-ray-defined temperature factors. Corresponding r.m.s. fluctuations for backbone atoms were 0.56 A by molecular dynamics and 0.49 A by X-ray. On the basis of these molecular dynamics studies of the isolated molecule, it is shown that amino acid residues corresponding to intermolecular contact sites of the crystalline enzyme are associated with high amplitude motion. All eight segments of a-helix and eight regions of /?-strand were well preserved except for unwinding of the five C-terminal residues of the a-helix 112-122 that form part of an intermolecular contact in the crystal. Four regions of P-strand and one a-helix with residues adjacent to or in t'he active site constitute a core of constant secondary structure and are shown not to change in relative orientation to each other during the course of the trajectory. The absence of the zinc ion does not markedly influence the stereochemical relationships of active site residues in the dynamically averaged protein. The extent of motional fluctuations of each of the subsites of substrate recognition in the active site has been evaluated. Active site residues responsible for specificity of substrate binding or splitting of the scissile bond exhibit low simulated motion. In contrast, residues in more distal sites of substrate recognition exhibit markedly greater motional fluctuations. This differential extent of dynamical motion is related to structural requirements of substrate hydrolysis.
Catalytic conformation of carboxypeptidase A
Journal of Molecular Biology, 1983
The structure of the mixed anhydride; acyl-enzyme intermediate of the rsterolptic reaction of carboxypeptidase A-is characterized by applicat,ion of cryoenzymologic. magnetic resonance. and molecular graphics methods with use of the Co2'substituted enzyme and the specific spin-label ester substrate O-3-(2,2.;i.h-tet,ramcthylpyrrolinyl-1-oxyl)-propen-2-oyl-I,-fl-phrnyllactate. A radial separation of 7.7 pi between the active site Co'+ and the nitroxide group in the low trmperaturestabilized acyl-enzyme intermediate is determined on the basis of their spin-spin (dipole ~dipole) interactions. Application of molecular graphics techniques shows that the only configuration of the substrate t'hat is strrically accommodated bv the active site vields a calculated metal ion-to-nit'roxide distance of 7.8 !I. iteric accommodation of the spin-label in the activr site requires severe torsional distortion around the aliphatic double bond of t#he propenoyl side-chain. Examirlation of the structure of the enzyme: spin-label intermediate reveals that thr distort'ion arises from steric interactions of t'he pyrrolinyl group with the protein at a position that corresponds to the site occupied by the penultimate amide residue of at1 oligopeptide substrate from the site of cleavage. Together wit'h kinetic data showing that, hydrolysis of the spin-label is governed by rat,e-limiting deacylation, the results indicate that geometric distortion of substratts by secondary interactions with thr t~rlzynlt'. in general. is an obligat~ory part of the caat,alytir action of c.arl)ox?-l)er~tidase A. LVhen viowetl with resf)ect to requirements for stereoelertronie control of bond cleavage in tetrahedral adducts of esters and amides (Deslongchamps. 197,5) the results suggest that torsional distortion during c*at)al>-sis results in rotation around t,he sc>issile bond of t,hc, sul)stratt>. and t,hat this rotation is required to form the mixed anhydride reaction intermediate. Thtw findings further support t)he interpretation that thus hydrolysis of esters and amides (aatalyzrd by ~arl)oxvf)el)t'idas~, A proceeds ac*c.ording to similar mechanisms except that formation of the rnixrd anhydride is r;rte-dt,t,c,l,rrti,ritlg in I)el)tide h,vdrolysis whik drac*ylat,ion of the mixed anh,vdridr is r,atc%-limiting in ester, hydrolysis. Additionally. in this study application of thr extension of the theory of the Solomon-Bloembergen-Morgan rquat,ions derived by Lindner (1965) for paramagnetic metal ions with 8 2 1 demonstrates that the zero-field splitting of th(x high-spin Co2' m the Ineta]-sui)stitutc,d enzytne has no significant influence in determination of the relaxation enhancement of solvent prot,ons by the active sitch metal ion. 6% 1,. (". Kc-0 ET .-I 1.
Proceedings of The National Academy of Sciences, 1993
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the key first step in photosynthetic CO_2 fixation, the reaction that incorporates CO_2 into sugar. In this study, refined crystal structures of unactivated tobacco RuBisCO and activated RuBisCO from spinach and tobacco, in complex with the reaction-intermediate analog 2-carboxyarabinitol 1,5-bisphosphate (CABP), are compared. Both plant enzymes are hexadecameric complexes of eight large and eight small subunits with a total relative molecular mass of ≈550,000. The comparison of activated and unactivated forms of RuBisCO provides insight into the dynamics of action of this enzyme. The catalytic site, which is open to the solvent in the unactivated enzyme, becomes shielded in the activated CABP complex. This shielding is accomplished by a 12-Å movement of the active-site "loop 6" (residues 331-338) and a disorder-order transition of three loops near the active-site entrance, the N terminus, the C terminus, and a loop comprising residues 64-68. All these residues belong to the catalytic large subunit. Domain rotations of about 2^circ are observed, also tightening the active-site cleft. These observations provide an explanation for the extremely tight binding (K_d <= 10-11 M) of the CABP molecule. A striking correlation exists between crystallographic temperature factors in the activated enzyme and the magnitude of the atomic movement upon activation.
Biochemistry, 1997
The interaction of native and Co(II)-substituted isozymes I and II of carbonic anhydrase (CA) with histamine, a well-known activator, was investigated kinetically, spectroscopically, and X-ray crystallographically. This activator is of the noncompetitive type with 4-nitrophenyl acetate and CO 2 as substrates for both HCA I and HCA II. The electronic spectrum of the adduct of Co(II)-HCA II with histamine is similar to the spectrum of the Co(II)-HCA II-phenol adduct, being only slightly different from that of the uncomplexed enzyme. This is the first spectroscopic evidence that the activator molecule binds within the active site, but not directly to the metal ion. X-ray crystallographic data for the adduct of HCA II with histamine showed that the activator molecule is bound at the entrance of the active site cavity in a position where it may actively participate in shuttling protons between the active site and the bulk solvent. The role of the activators and the reported X-ray crystal structure of the HCA II-histamine adduct has prompted us to reexamine the X-ray structures of the different CA isozymes in order to find a structural basis accounting for their large differences in catalytic rate. A tentative explanation is proposed on the basis of possible pathways of proton transfer, which constitute the rate-limiting step in the catalytic reaction. † This research was financed by European Union Grant ERBCIPDCT 940051 and by Consiglio Nazionale delle Ricerche Grant 96.01270.PF37. ‡ The structure of the HCA II-histamine adduct has been deposited in the Brookhaven Protein Data Bank, accession code 4TST.