Water chain formation and possible proton pumping routes in Rhodobacter sphaeroides cytochrome c oxidase: a molecular dynamics comparison of the wild type and R481K mutant

Cytochrome c oxidase (CcO) converts the energy from redox and oxygen chemistry to support proton translocation and create a transmembrane DeltamuH(+) used for ATP production. Molecular dynamics (MD) simulations were carried out to probe for the formation water chains capable of participating in proton translocation. Attention was focused on the region between and above the a and a(3) hemes where well-defined water chains have not been identified in crystallographic studies. An arginine (R481) (Rhodobacter sphaeroides numbering), positioned between the D-propionates of the hemes, had been mutated in vivo to lysine and showed to have altered activity consistent with an altered proton conductance [Qian, J., Mills, D. A., Geren, L., Wang, K. F., Hoganson, C. W., Schmidt, B., Hiser, C., Babcock, G. T., Durham, B., Millett, F., and Ferguson-Miller, S. (2004) Role of the conserved arginine pair in proton and electron transfer in cytochrome c oxidase, Biochemistry 43, 5748-5756; also see the accompanying paper by Mills et al.]. This mutant was created in silico, and the MD results for the mutant and wild type were compared to explore the effects on the formation of hydrogen-bonded water chains by this mutation. The simulations reveal the presence of hydrogen-bonded water chains that lead from E286 through the region above the hemes to the Mg(2+), and from E286 to the heme a(3) D-propionate and the binuclear center. The R481K mutant does not form as many, or as extensive, water chains as wild-type CcO, due to a new conformation of residues in a large loop between helices III and IV in subunit I, indicating a reduction in the level of water chain formation in the mutant. This loop appears to play a role in controlling the formation of hydrogen-bonded water chains above the hemes. The results suggest a possible gating mechanism for proton movement that includes key residues W172 and Y175 on the loop and F282 on helix VI.     

Molecular modeling of the ternary complex of Rusticyanin-cytochrome c4-cytochrome oxidase: an insight to possible H-bond mediated recognition and electron transfer reaction in T. ferrooxidans

The metabolism of Thiobacillus ferrooxidans involves electron transfer from the Fe+2 ions in the extracellular environment to the terminal oxygen in the bacterial cytoplasm through a series of periplasmic proteins like Rusticyanin (RCy), Cytochrome (Cyt c4), and Cytochrome oxidase (CcO). The energy minimization and MD studies reveal the stabilization of the three redox proteins in their ternary complex through the direct and water mediated H-bonds and electrostatic interaction. The surface exposed polar residues of the three proteins, i.e., RCy (His 143, Thr 146, Lys 81, Glu 20), Cyt c4 (Asp 5, 15, 52, Ser 14, Glu 61), and CcO (Asp 135, Glu 126, 140, 142, Thr 177) formed the intermolecular hydrogen bonds and stabilized the ternary complex. The oxygen (Oepsilon1) of Glu 126, 140, and 142 on subunit II of the CcO interact to the exposed side-chain and Ob atoms of the Asp 52 of Cyt c4 and Glu 20 and Leu 12 of RCy. The Asp 135 of subunit II also forms H-bond with the Nepsilon atom of Lys 81 of RCy. The Oepsilon1 of Glu 61 of Cyt c4 is also H-bonded to Ogamma atom of Thr 177 of CcO. Solvation followed by MD studies of the ternary protein complex revealed the presence of seven water molecules in the interfacial region of the interacting proteins. Three of the seven water molecules (W 79, W 437, and W 606) bridged the three proteins by forming the hydrogen bonded network (with the distances approximately 2.10-2.95 A) between the Lys 81 (RCy), Glu 61 (Cyt c4), and Asp 135 (CcO). Another water molecule W 603 was H-bonded to Tyr 122 (CcO) and interconnected the Lys 81 (RCy) and Asp 135 (CcO) through the water molecules W 606 and W 437. The other two water molecules (W 21 and W 455) bridged the RCy to Cyt c4 through H-bonds, whereas the remaining W 76 interconnected the His 53 (Cytc4) to Glu 126 (CcO) with distances approximately 2.95-3.0 A.     

Plasticity of proton pathway structure and water coordination in cytochrome c oxidase

Cytochrome c oxidase (CytcO) is a redox-driven, membrane-bound proton pump. One of the proton transfer pathways of the enzyme, the D pathway, used for the transfer of both substrate and pumped protons, accommodates a network of hydrogen-bonded water molecules that span the distance between an aspartate (Asp(132)), near the protein surface, and glutamate Glu(286), which is an internal proton donor to the catalytic site. To investigate how changes in the environment around Glu(286) affect the mechanism of proton transfer through the pathway, we introduced a non-hydrogen-bonding (Ala) or an acidic residue (Asp) at position Ser(197) (S197A or S197D), located approximately 7 A from Glu(286). Although Ser(197) is hydrogen-bonded to a water molecule that is part of the D pathway "proton wire," replacement of the Ser by an Ala did not affect the proton transfer rate. In contrast, the S197D mutant CytcO displayed a turnover activity of approximately 35% of that of the wild-type CytcO, and the O(2) reduction reaction was not linked to proton pumping. Instead, a fraction of the substrate protons was taken from the positive ("incorrect") side of the membrane. Furthermore, the pH dependence of the proton transfer rate was altered in the mutant CytcO. The results indicate that there is plasticity in the water coordination of the proton pathway, but alteration of the electrostatic potential within the pathway results in uncoupling of the proton translocation machinery.     

Molecular Modeling of the Ternary Complex of Rusticyanin-Cytochrome c4-Cytochrome Oxidase: An Insight to Possible H-Bond Mediated Recognition and Electron Transfer Reaction in T.ferrooxidans

Abstract

The metabolism of Thiobacillus ferrooxidans involves electron transfer from the Fe⁺² ions in the extracellular environment to the terminal oxygen in the bacterial cytoplasm through a series of periplasmic proteins like Rusticyanin (RCy), Cytochrome (Cyt c₄), and Cytochrome oxidase (CcO). The energy minimization and MD studies reveal the stabilization of the three redox proteins in their ternary complex through the direct and water mediated H-bonds and electrostatic interaction. The surface exposed polar residues of the three proteins, i.e., RCy (His 143, Thr 146, Lys 81, Glu 20), Cyt c₄ (Asp 5, 15, 52, Ser 14, Glu 61), and CcO (Asp 135, Glu 126, 140, 142, Thr 177) formed the intermolecular hydrogen bonds and stabilized the ternary complex. The oxygen (O^εl) of Glu 126, 140, and 142 on subunit II of the CcO interact to the exposed side-chain and O^b atoms of the Asp 52 of Cyt c₄ and Glu 20 and Leu 12 of RCy. The Asp 135 of subunit II also forms H-bond with the N^ε atom of Lys 81 of RCy. The O^εl of Glu 61 of Cyt c₄ is also H-bonded to O^γ atom of Thr 177 of CcO. Solvation followed by MD studies of the ternary protein complex revealed the presence of seven water molecules in the interfacial region of the interacting proteins. Three of the seven water molecules (W 79, W 437, and W 606) bridged the three proteins by forming the hydrogen bonded network (with the distances ～ 2.10–2.95 Å) between the Lys 81 (RCy), Glu 61 (Cyt c₄), and Asp 135 (CcO). Another water molecule W 603 was H-bonded to Tyr 122 (CcO) and interconnected the Lys 81 (RCy) and Asp 135 (CcO) through the water molecules W 606 and W 437. The other two water molecules (W 21 and W 455) bridged the RCy to Cyt c₄ through H-bonds, whereas the remaining W 76 interconnected the His 53 (Cytc₄) to Glu 126 (CcO) with distances ～ 2.95–3.0 Å.     

Exploration of the cytochrome c oxidase pathway puzzle and examination of the origin of elusive mutational effects

Gaining detailed understanding of the energetics of the proton-pumping process in cytochrome c oxidase (CcO) is a problem of great current interest. Despite promising mechanistic proposals, so far, a physically consistent model that would reproduce all the relevant barriers needed to create a working pump has not been presented. In addition, there are major problems in elucidating the origin of key mutational effects and in understanding the nature of the apparent pK(a) values associated with the pH dependencies of specific proton transfer (PT) reactions in CcO. This work takes a key step in resolving the above problems, by considering mutations, such as the Asn139Asp replacement, that blocks proton pumping without affecting PT to the catalytic site. We first introduce a formulation that makes it possible to relate the apparent pK(a) of Glu286 to different conformational states of this residue. We then use the new formulation along with the calculated pK(a) values of Glu286 at these different conformations to reproduce the experimentally observed apparent pK(a) of the residue. Next, we take the X-ray structures of the native and Asn139Asp mutant of the Paracoccus denitrificans CcO (N131D in this system) and reproduce for the first time the change in the primary PT pathways (and other key features) based on simulations that start with the observed structural changes. We also consider the competition between proton transport to the catalytic site and the pump site, as a function of the bulk pH, as well as the H/D isotope effect, and use this information to explore the relative height of the two barriers. The paper emphasizes the crucial role of energy-based considerations that include the PT process, and the delicate control of PT in CcO.     

Network analysis of a proposed exit pathway for protons to the P-side of cytochrome c oxidase

Cytochrome c Oxidase (CcO) reduces O₂, the terminal electron acceptor, to water in the aerobic, respiratory electron transport chain. The energy released by O₂ reductions is stored by removing eight protons from the high pH, N-side, of the membrane with four used for chemistry in the active site and four pumped to the low pH, P-side. The proton transfers must occur along controllable proton pathways that prevent energy dissipating movement towards the N-side. The CcO N-side has well established D- and K-channels to deliver protons to the protein interior. The P-side has a buried core of hydrogen-bonded protonatable residues designated the Proton Loading Site cluster (PLS cluster) and many protonatable residues on the P-side surface, providing no obvious unique exit. Hydrogen bond pathways were identified in Molecular Dynamics (MD) trajectories of Rb. sphaeroides CcO prepared in the P_R state with the heme a₃ propionate and Glu286 in different protonation states. Grand Canonical Monte Carlo sampling of water locations, polar proton positions and residue protonation states in trajectory snapshots identify a limited number of water mediated, proton paths from PLS cluster to the surface via a (P-exit) cluster of residues. Key P-exit residues include His93, Ser168, Thr100 and Asn96. The hydrogen bonds between PLS cluster and P-exit clusters are mediated by a water wire in a cavity centered near Thr100, whose hydration can be interrupted by a hydrophobic pair, Leu255B (near Cu_A) and Ile99. Connections between the D channel and PLS via Glu286 are controlled by a second, variably hydrated cavity.

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Metabolism of some amino acids in relation to the photorespiratory nitrogen cycle of pea leaves

¹⁵N-labelled (amino group) asparagine (Asn), glutamate (Glu), alanine (Ala), aspartate (Asp) and serine (Ser) were used to study the metabolic role and the participation of each compound in the photorespiratory N cycle ofPisum sativum L. leaves. Asparagine was utilised as a nitrogen source by either deamidation or transamination, Glu was converted to Gln through NH₃ assimilation and was a major amino donor for transamination, and Ala was utilised by transamination to a range of amino acids. Transamination also provided a pathway for Asp utilisation, although Asp was also used as a substrate for Asn synthesis. In the photorespiratory synthesis of glycine (Gly), Ser, Ala, Glu and Asn acted as sources of amino-N, contributing, in the order given, 38, 28, 23, and 7% of the N for glycine synthesis; Asp provided less than 4% of the amino-N in glycine. Calculations based on the incorporation of¹⁵N into Gly indicated that about 60% (Ser), 20% (Ala), 12% (Glu) and 11% (Asn) of the N metabolised from each amino acid was utilised in the photorespiratory nitrogen cycle.Abbreviations Ala alamine - Asn asparagine - Asp aspartate - Glu glutamate - MOA methoxylamine - Ser serine     

Structure-function relationships of Na(+), K(+), ATP, or Mg(2+) binding and energy transduction in Na,K-ATPase

The focus of this article is on progress in establishing structure-function relationships through site-directed mutagenesis and direct binding assay of Tl(+), Rb(+), K(+), Na(+), Mg(2+) or free ATP at equilibrium in Na,K-ATPase. Direct binding may identify residues coordinating cations in the E(2)[2K] or E(1)P[3Na] forms of the ping-pong reaction sequence and allow estimates of their contributions to the change of Gibbs free energy of binding. This is required to understand the molecular basis for the pronounced Na/K selectivity at the cytoplasmic and extracellular surfaces. Intramembrane Glu(327) in transmembrane segment M4, Glu(779) in M5, Asp(804) and Asp(808) in M6 are essential for tight binding of K(+) and Na(+). Asn(324) and Glu(327) in M4, Thr(774), Asn(776), and Glu(779) in 771-YTLTSNIPEITP of M5 contribute to Na(+)/K(+) selectivity. Free ATP binding identifies Arg(544) as essential for high affinity binding of ATP or ADP. In the 708-TGDGVND segment, mutations of Asp(710) or Asn(713) do not interfere with free ATP binding. Asp(710) is essential and Asn(713) is important for coordination of Mg(2+) in the E(1)P[3Na] complex, but they do not contribute to Mg(2+) binding in the E(2)P-ouabain complex. Transition to the E(2)P form involves a shift of Mg(2+) coordination away from Asp(710) and Asn(713) and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp(369).     

A molecular dynamics study of water chain formation in the proton-conducting K channel of cytochrome c oxidase

The formation of water chains in cytochrome c oxidase (CcO) is studied by molecular dynamics (MD). Focus is on water chains in the K channel that can supply a proton to the binuclear center (the heme a3 Fe/CuB region), the site of O2 reduction. By assessing the presence of chains of any length on a short time scale (0.1 ps), a view of the kinds of chains and their persistence is obtained. Chains from the entry of the channel on the inner membrane to Thr359 (Rhodobacter sphaeroides numbering) are often present but are blocked at that point until a rotation of the Thr359 side chain occurs, permitting formation of chains from Thr359 towards the binuclear center. No continuous hydrogen-bonded water chains are found connecting Thr359 and the binuclear center. Instead, waters hydrogen bond from Thr359 to the hydroxyl of the heme a3 farnesyl and then continue to the binuclear center via Tyr288, which has been identified as a source of a proton for O2 reduction. Three hydrogen-bonded waters are found to be present in the binuclear center after a sufficiently long simulation time. One is ligated to the CuB and could be associated with a water (or hydroxyl) identified in the crystal structure as the fourth ligand of CuB. The water hydrogen-bonded to the hydroxyl of Tyr288 is extremely persistent and well positioned to participate in O2 reduction. The third water is located where O2 is often suggested to reside in mechanistic studies of O2 reduction.     

Stabilization of apoflavodoxin by replacing hydrogen-bonded charged Asp or Glu residues by the neutral isosteric Asn or Gln

Knowledge of protein stability principles provides a means to increase protein stability in a rational way. Here we explore the feasibility of stabilizing proteins by replacing solvent-exposed hydrogen-bonded charged Asp or Glu residues by the neutral isosteric Asn or GLN: The rationale behind this is a previous observation that, in some cases, neutral hydrogen bonds may be more stable that charged ones. We identified, in the apoflavodoxin from Anabaena PCC 7119, three surface-exposed aspartate or glutamate residues involved in hydrogen bonding with a single partner and we mutated them to asparagine or glutamine, respectively. The effect of the mutations on apoflavodoxin stability was measured by both urea and temperature denaturation. We observed that the three mutant proteins are more stable than wild-type (on average 0.43 kcal/mol from urea denaturation and 2.8 degrees C from a two-state analysis of fluorescence thermal unfolding data). At high ionic strength, where potential electrostatic repulsions in the acidic apoflavodoxin should be masked, the three mutants are similarly more stable (on average 0.46 kcal/mol). To rule out further that the stabilization observed is due to removal of electrostatic repulsions in apoflavodoxin upon mutation, we analysed three control mutants and showed that, when the charged residue mutated to a neutral one is not hydrogen bonded, there is no general stabilizing effect. Replacing hydrogen-bonded charged Asp or Glu residues by Asn or Gln, respectively, could be a straightforward strategy to increase protein stability.     

Alternative initial proton acceptors for the D pathway of Rhodobacter sphaeroides cytochrome c oxidase

To characterize protein structures that control proton uptake, we assayed forms of cytochrome c oxidase (CcO) containing a carboxyl or a thiol group in line with the initial, internal waters of the D pathway for proton transfer in the presence and absence of subunit III. Subunit III provides approximately half of the protein surrounding the entry region of the D pathway. The N139D/D132N mutant contains a carboxyl group 6 ? within the D pathway and lacks the normal, surface-exposed proton acceptor, Asp-132. With subunit III, the steady-state activity of this mutant is slow, but once subunit III is removed, its activity is the same as that of wild-type CcO lacking subunit III (～1800 H+/s). Thus, a carboxyl group～25% within the pathway enhances proton uptake even though the carboxyl has no direct contact with bulk solvent. Protons from solvent apparently move to internal Asp-139 through a short file of waters, normally blocked by subunit III. Cys-139 also supports rapid steady-state proton uptake, demonstrating that an anion other than a carboxyl can attract and transfer protons into the D pathway. When both Asp-132 and Asp/Cys-139 are present, the removal of subunit III increases CcO activity to rates greater than that of normal CcO because of simultaneous proton uptake by two initial acceptors. The results show how the environment of the initial proton acceptor for the D pathway in these CcO forms dictates the pH range of CcO activity, with implications for the function of Asp-132, the normal proton acceptor.     

Structurally conserved water molecules in ribonuclease T1

In the high resolution (1.7-1.9 A) crystal structures of ribonuclease T1 (RNase T1) in complex with guanosine, guanosine 2'-phosphate, guanylyl 2',5'-guanosine, and vanadate, there are 30 water sites in nearly identical (+/- 1 A) positions that are considered conserved. One water is tightly bound to Asp76(O delta), Thr93(O gamma), Cys6(O), and Asn9(N); another bridges two loops by hydrogen-bonding to Tyr68(O eta) and to Ser35(N), Asn36(N); a loop structure is stabilized by two waters coordinated to Gly31(O) and His27(N delta), and by water bound to cis-Pro39(O). Most notable is a hydrogen-bonded chain of 10 water molecules. Waters 1-5 of this chain are inaccessible to solvent, are anchored at Trp59(N), and stitch together the loop formed by segments 60-68; waters 5-8 coordinate to Ca2+, and waters 9 and 10 hydrogen-bond to N-terminal side chains of the alpha-helix. The water chain and two conserved water molecules are bound to amino acids adjacent to the active site residues His40, Glu58, Arg77, and His92; they are probably involved in maintaining their spatial orientation required for catalysis. Water sites must be considered in genetic engineering; the mutation Trp59Tyr, which probably influences the 10-water chain, doubles the catalytic activity of RNase T1.     

Role of Asn(2) and Glu(7) residues in the oxidative folding and on the conformation of the N-terminal loop of apamin

The X-ray structure of [N-acetyl]-apamin has been solved at 0.95 A resolution. It consists of an 1-7 N-terminal loop stabilized by an Asn-beta-turn motif (2-5 residues) and a helical structure spanning the 9-18 residues tightly linked together by two disulfide bonds. However, neither this accurate X-ray nor the available solution structures allowed us to rationally explain the unusual downfield shifts observed for the Asn(2) and Glu(7) amide signals upon Glu(7) carboxylic group ionization. Thus, apamin and its [N-acetyl], [Glu(7)Gln], [Glu(7)Asp], and [Asn(2)Abu] analogues and submitted to NMR structural studies as a function of pH. We first demonstrated that the Glu(7) carboxylate group is responsible for the large downfield shifts of the Asn(2) and Glu(7) amide signals. Then, molecular dynamics (MD) simulations suggested unexpected interactions between the carboxylate group and the Asn(2) and Glu(7) amide protons as well as the N-terminal alpha-amino group, through subtle conformational changes that do not alter the global fold of apamin. In addition, a structural study of the [Asn(2)Abu] analogue, revealed an essential role of Asn(2) in the beta-turn stability and the cis/trans isomerization of the Ala(5)-Pro(6) amide bond. Interestingly, this proline isomerization was shown to also depend on the ionization state of the Glu(7) carboxyl group. However, neither destabilization of the beta-turn nor proline isomerization drastically altered the helical structure that contains the residues essential for binding. Altogether, the Asn(2) and Glu(7) residues appeared essential for the N-terminal loop conformation and thus for the selective formation of the native disulfide bonds but not for the activity.     

Crystallographic evidence for Tyr 157 functioning as the active site base in human UDP-galactose 4-epimerase

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-glucose and UDP-galactose during normal galactose metabolism. In humans, deficiencies in this enzyme lead to the complex disorder referred to as epimerase-deficiency galactosemia. Here, we describe the high-resolution X-ray crystallographic structures of human epimerase in the resting state (i.e., with bound NAD(+)) and in a ternary complex with bound NADH and UDP-glucose. Those amino acid side chains responsible for anchoring the NAD(+) to the protein include Asp 33, Asn 37, Asp 66, Tyr 157, and Lys 161. The glucosyl group of the substrate is bound to the protein via the side-chain carboxamide groups of Asn 187 and Asn 207. Additionally, O(gamma) of Ser 132 and O(eta) of Tyr 157 lie within 2.4 and 3.1 A, respectively, of the 4'-hydroxyl group of the sugar. Comparison of the polypeptide chains for the resting enzyme and for the protein with bound NADH and UDP-glucose demonstrates that the major conformational changes which occur upon substrate binding are limited primarily to the regions defined by Glu 199 to Asp 240 and Gly 274 to Tyr 308. Additionally, this investigation reveals for the first time that a conserved tyrosine, namely Tyr 157, is in the proper position to interact directly with the 4'-hydroxyl group of the sugar substrate and to thus serve as the active-site base. A low barrier hydrogen bond between the 4'-hydroxyl group of the sugar and O(gamma) of Ser 132 facilitates proton transfer from the sugar 4'-hydroxyl group to O(eta) of Tyr 157.     

Aspzincin, a family of metalloendopeptidases with a new zinc-binding motif. Identification of new zinc-binding sites (His(128), His(132), and Asp(164)) and three catalytically crucial residues (Glu(129), Asp(143), and Tyr(106)) of deuterolysin from Aspergillus oryzae by site-directed mutagenesis.

Deuterolysin (EC 3.4.24.39; formerly designated as neutral proteinase II) from Aspergillus oryzae, which contains 1 g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. Active-site determination of the recombinant enzyme expressed in Escherichia coli was performed by site-directed mutagenesis. Substitutions of His(128) and His(132) with Arg, of Glu(129) with Gln or Asp, of Asp(143) with Asn or Glu, of Asp(164) with Asn, and of Tyr(106) with Phe resulted in almost complete loss of the activity of the mutant enzymes. It can be concluded that His(128), His(132), and Asp(164) provide the Zn(2+) ligands of the enzyme according to a (65)Zn binding assay. Based on site-directed mutagenesis experiments, it was demonstrated that the three essential amino acid residues Glu(129), Asp(143), and Tyr(106) are catalytically crucial residues in the enzyme. Glu(129) may be implicated in a central role in the catalytic function. We conclude that deuterolysin is a member of a family of Zn(2+) metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH + D" motif and an aspartic acid as the third zinc ligand.     

Activation of factor V during intrinsic and extrinsic coagulation. Inhibition by heparin, hirudin and D-Phe-Pro-Arg-Ch2Cl.

Ser130, Asp131 and Asn132 ('SDN') are highly conserved residues in class A beta-lactamases forming one wall of the active-site cavity. All three residues of the SDN loop in Streptomyces albus G beta-lactamase were modified by site-directed mutagenesis. The mutant proteins were expressed in Streptomyces lividans, purified from culture supernatants and their kinetic parameters were determined for several substrates. Ser130 was substituted by Asn, Ala and Gly. The first modification yielded an almost totally inactive protein, whereas the smaller-side-chain mutants (A and G) retained some activity, but were less stable than the wild-type enzyme. Ser130 might thus be involved in maintaining the structure of the active-site cavity. Mutations of Asp131 into Glu and Gly proved to be highly detrimental to enzyme stability, reflecting significant structural perturbations. Mutation of Asn132 into Ala resulted in a dramatically decreased enzymic activity (more than 100-fold) especially toward cephalosporin substrates, kcat. being the most affected parameter, which would indicate a role of Asn132 in transition-state stabilization rather than in ground-state binding. Comparison of the N132A and the previously described N132S mutant enzymes underline the importance of an H-bond-forming residue at position 132 for the catalytic process.     

Thermodynamic and mutational studies of l-N-carbamoylase from Sinorhizobium meliloti CECT 4114 catalytic centre

Purified site-directed mutants of Sinorhizobium meliloti CECT 4114 l-N-carbamoylase (SmLcar) in which Glu132, His230, Asn279 and Arg292 were replaced have been studied by kinetic methods and isothermal titration calorimetry (ITC). The importance of His230, Asn279 and Arg292 residues in the recognition of N-carbamoyl-l-alpha-amino acids has been proved. The role of Glu132 has been confirmed in substrate hydrolysis. ITC has confirmed two Ni atoms per monomer of wild type enzyme, and two equal and independent substrate binding sites (one per monomer). Homology modelling of SmLcar supports the importance of His87, His194, His386, Glu133 and Asp98 in metal binding. A comprehensive reaction mechanism is proposed on the basis of binding experiments measured by ITC, kinetic assays, and homology of the active centre with beta-alanine synthase from Saccharomyces kluyveri and other enzymes.