Residues In Enzyme Active Site Research Articles

Direct Identification of Enzyme Active Site Residues by Solid-State REDOR NMR: Application to KDO8P Synthase

In this communication we report the first direct identification of active site residues of the enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS). This is accomplished by the application of rotational-echo double-resonance1 (REDOR) solid-state NMR experiments to lyophilized binary complexes of uniformly 15N-labeled KDO8PS with each of its natural substrates, phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate (A5P). Its complex with the mechanism-based inhibitor, 2 (Scheme 1), the most potent inhibitor known to date2 (Ki ) 0.4 μM), is studied as well and its binding is compared to that of the natural substrates. The REDOR technique1 is based on selective reintroduction of (magnetic) dipolar interactions between pairs of specific types of nuclei, and in favorable cases enables their accurate measurement from which distance constraints can be obtained.3 Its application for the study of protein-ligand interactions is achieved by incorporation of stable isotopes at known positions in the protein that are proximate to (labeled) ligand moieties. In previous studies, REDOR measured distances were shown to be in excellent agreement with X-ray data.3a,b Moreover, 31P{13C} REDOR was employed to investigate the conformation of the bound inhibitor glyphosate in the enzyme-substrate-inhibitor ternary complex of 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS),3c for which no X-ray structure is available. REDOR, e.g., 31P{13C} and 15N {31P}, was also used for the determination of structural constraints in the above ternary complex,3d and to directly monitor transient enzyme-intermediate complexes of EPSPS3e and of UDP-GlcNac enolpyruvoyl transferase (Mur Z).3f The enzyme KDO8PS catalyzes the condensation reaction between A5P and PEP to form KDO8P and inorganic phosphate (Pi, Scheme 1).4 This important enzymatic reaction plays a crucial role in the assembly process of lipopolysaccharides of most Gramnegative bacteria,5 and is therefore an attractive target for the design of novel antibacterial drugs. Interestingly, unlike most PEPutilizing enzymes that cleave the high-energy P-O bond of PEP, KDO8PS and its family members (Mur Z, EPSPS, and 3-deoxyD-arabino-heptulosonate-7-phosphate synthase6 (DAHPS)) cleave the C-O bond of PEP.7 While the mechanisms of EPSPS8a (targeted by the herbicide glyphosate) and Mur Z8b (targeted by the antibiotic fosfomycin) were unambiguously characterized, the mechanisms of KDO8PS and DAHPS continue to be uncertain. Recent studies9a,2 on KDO8PS, including the synthesis and evaluation of the first acyclic bisubstrate inhibitor (2),2 supported a mechanism involving the formation of an acyclic bisphosphate intermediate 1 (Scheme 1).9b Mutagenesis studies10 identified in E. coli KDO8PS two vital cysteines, Cys38 and Cys166, yet their specific role still remains unclear. In addition, since to date neither the crystal structure nor the identity of the active site residues of KDO8PS are known, rational design of potent inhibitors for this enzyme is impaired. As part of our study on the structurefunction relationship of KDO8PS, [u-15N]KDO8PS was prepared and interactions between 15N enzyme labels to 31P nuclei of the ligands were examined by REDOR NMR.11 The 15N NMR CPMAS echo spectrum of [u-15N]KDO8PS binary complex with PEP (Figure 1a) shows resolved peaks for the nitrogen atoms of 10 side-chain arginine (guanidino: internal , 60 ppm; terminal η, 49 ppm), 22 lysine ( 9.0 ppm) residues, and an intense amide peak (95 ppm). Peaks of six side-chain histidines (δ1 224; 2 143 ppm) are not shown. In addition, detailed inspection of this spectrum reveals fine structure for the arginine (56.0 ppm), and a high field shoulder for lysine (3.0 ppm), attributed to a single arginine and to a single lysine residue residing in a distinct chemical environment. Interestingly, these two peaks are also present in the 15N CPMAS reference spectra of the other two complexes, KDO8PS-A5P and KDO8PS-2 (spectra not shown), implying that they arise from chemically distinct environments inherent to the enzyme itself, and are therefore not a result of interaction with a specific ligand. As will be shown below, these residues will be associated with specific recognition and binding of PEP by the enzyme.

Different Types of Interactions Involving Cysteine Sulfhydryl Group in Proteins

Various types of interactions involving the sulfhydryl group of free cysteine residues have been analyzed using known protein structures. In a hydrogen bond the -SH group is more amenable to donating its proton to a carbonyl group, rather than acting as a proton acceptor. It rarely interacts with a carboxylate group, and is a poor ligand to bind an anionic substrate. It is quite prone to make contacts that are definitely non-hydrogen bond type. In the S…C=0 interaction the S atom is placed on the face of an amide group (mostly from the main-chain, but there are cases from the side-chain also) close to the C atom. Cases of S…N interaction, where the S atom is on top of the N atom of another residue (both main-, as well as side-chains, including the guanidinium group) are also observed. A considerable number of Cys residues have aromatic residues as neighbors, and here too, the preferred mode of interaction is along the face. The intra-residue S…C=0 interaction constrains the main-chain and side-chain torsion angles (ψ and x1), whereas the inter-residue interactions are non-local and stabilize the tertiary structure. The S…C=0 interaction may have a role in lowering the pK avalues of the Cys residues in enzyme active sites.

Kinetic and Mechanistic Characterization of a Mammalian Protein-tyrosine Phosphatase, PTP1

The kinetic mechanism of the hydrolysis of phosphate monoesters catalyzed by a soluble form of rat protein-tyrosine phosphatase (PTPase), PTP1, was probed with a variety of steady-state and pre-steady-state kinetic techniques. Product inhibition and 18O exchange experiments are consistent with the enzymatic reaction proceeding through two chemical steps, i.e. formation and breakdown of a covalent phosphoenzyme intermediate. The variation of kcat/Km with pH indicates that three ionizable groups are involved in enzyme substrate binding and catalysis. The first group must be deprotonated and is attributed to the second ionization of the substrate. The other two groups with pK alpha values of 5.1 and 5.5 correspond to two enzyme active site residues. The kcat-pH profiles for both p-nitrophenyl phosphate and beta-naphthyl phosphate are bell-shaped and are superimposable, with the apparent pK alpha values derived from the acidic limb and the basic limb of the profile being 4.4 and 6.8, respectively. This suggests that the rate-limiting step corresponds to the decomposition of the phosphoenzyme intermediate at all pH values. Results from leaving group dependence of kcat at two different pH values support the above conclusion. Furthermore, burst kinetics have been demonstrated with PTP1 using p-nitrophenyl phosphate as a substrate. The rate constants for the formation and the breakdown of the intermediate are 241 and 12 s-1, respectively, at pH 6.0 and 3.5 degrees C. A normal D2O solvent isotope effect (kcatH/kcatD = 1.5) is associated with the breakdown of the phosphoenzyme intermediate, indicating a solvent-derived proton in the transition state. The leaving group dependence of kcat/Km suggests that there is a strong electrophilic interaction between the enzyme and the leaving group oxygen in the transition state of the phosphorylation event. These results are compared with those of the Yersinia PTPase and suggest that the mechanism for PTPase-catalyzed phosphate monoester hydrolysis is conserved from bacterial to mammals.

COMPUTER SIMULATION OF STAPHYLOCOCCAL NUCLEASE ACTION ON THYMIDINE 3′,5′-BIS(PHOSPHATE) (pdTp)

Abstract Computer modeling based on a molecular mechanics approach was used to explore the mechanism of staphylococcal nuclease action on the thymidine 3′-phosphate 5′-(p-nitrophenyl phosphate)-calcium ion substrate complex. The active-site residues Asp-21, Asp-40, Glu-43, Arg-35, Tyr-113, Lys-84, Arg-87, and Tyr-85 were included in the calculation. Initially the X-ray coordinates were used for the enzyme active-site residues with the inhibitor substrate, thymidine 3′,5′-bis(phosphate) (pdTp), in place. Subsequently, introduction of the p-nitrophenyl group and energy minimization gave the coordinates for the active substrate. In-line attack by hydroxide was simulated by approach opposite the phosphoryl oxygen atom attached to the p-nitrophenyl group (path 1) and by approach opposite the phosphoryl oxygen atom attached to thymidine (path 2). The former pathway is the route that leads to products observed in nonenzymatic cleavage. The latter pathway is the enzymatic route. Computer simulation of the hydroly...

How do MgATP analogues differentially modify high‐affinity and low‐affinity ATP binding sites of Na+/K+ ‐ATPase?

The exchange-inert tetra-ammino-chromium complex of ATP [Cr(NH3)4ATP], unlike the analogous cobalt complex Co(NH3)4ATP, inactivated Na+/K(+)-ATPase slowly by interacting with the high-affinity ATP binding site. The inactivation proceeded at 37 degrees C with an inactivation rate constant of 1.34 x 10(-3) min-1 and with a dissociation constant of 0.62 microM. To assess the potential role of the water ligands of metal in binding and inactivation, a kinetic analysis of the inactivation of Na+/K(+)-ATPase by Cr(NH3)4ATP, and its H2O-substituted derivatives Cr(NH3)3(H2O)ATP, Cr(NH3)2(H2O)2ATP and Cr(H2O)4ATP was carried out. The substitution of the H2O ligands with NH3 ligands increased the apparent binding affinity and decreased the inactivation rate constants of the enzyme by these complexes. Inactivation by Cr(H2O)4ATP was 29-fold faster than the inactivation by Cr(NH3)4ATP. These results suggested that substitution to Cr(III) occurs during the inactivation of the enzyme. Additionally hydrogen bonding between water ligands of metal and the enzyme's active-site residues does not seem to play a significant role in the inactivation of Na+/K(+)-ATPase by Cr(III)-ATP complexes. Inactivation of the enzyme by Rh(H2O)nATP occurred by binding of this analogue to the high-affinity ATP site with an apparent dissociation constant of 1.8 microM. The observed inactivation rate constant of 2.11 x 10(-3) min-1 became higher when Na+ or Mg2+ or both were present. The presence of K+ however, increased the dissociation constant without altering the inactivation rate constant. High concentrations of Na+ reactivated the Rh(H2O)nATP-inactivated enzyme. Co(NH3)4ATP inactivates Na+/K(+)-ATPase by binding to the low-affinity ATP binding site only at high concentrations. However, inactivation of the enzyme by Cr(III)-ATP or Rh(III)-ATP complexes was prevented when low concentrations of Co(NH3)4ATP were present. This indicates that, although Co(NH3)4ATP interacts with both ATP sites, inactivation occurs only through the low-affinity ATP site. Inactivation of Na+/K(+)-ATPase was faster by the delta isomer of Co(NH3)4ATP than by the delta isomer. Co(NH3)4ATP, but not Cr(H2O)4ATP or adenosine 5'-[beta,gamma-methylene]triphosphate competitively inhibited K(+)-activated p-nitrophenylphosphatase activity of Na+/K(+)-ATPase, which is assumed to be a partial reaction of the enzyme catalyzed by the low-affinity ATP binding site.