Conformation of bound substrate

Conformation of Bound Substrate. Now the question of the conformation of bound DHF substrate was addressed. Simulations were run on DHFR/DHF/NADPH ternary complexes to investigate both the structural and energetic requirements associated with the reactive and nonreactive forms of DHF (see Figure 14). As above, the crystallographic information available for... 

The reaction proceeds via a ternary complex mechanism (CAT Ac-CoA Cm CAT CoA Ac-Cm) with a random order of addition of substrates (18). The progression from the binary complex to the ternary complex involves subtle changes in the structure of the enzyme and/or the conformations of bound substrates and accompanies a threefold decrease in affinity the fCj of Cm and Ac-CoA in respective binary complexes are 4 and 30 fxM, whereas the corresponding values of the ternary complex are 12 and 90 (iM, respectively (20). The RDS is thought to involve both product release and ternary complex interconversion. No evidence exists for the formation of an acetyl-enzyme intermediate. 

Substrate analogs which promise to be particularly good active-site probes are those which are conformationally restricted. One key feature of enzymatic processes is that when a substrate is bound to an enzyme, probably only one of the many possible conformations of the substrate molecule is assumed. Consequently, before a detailed mechanism for an enzymatic process can be formulated, the preferred conformations of each of the enzyme-bound substrates must be known. ... 

The mechanism and sequence of events that control delivery of protons and electrons to the FeMo cofactor during substrate reduction is not well understood in its particulars.8 It is believed that conformational change in MoFe-protein is necessary for electron transfer from the P-cluster to the M center (FeMoco) and that ATP hydrolysis and P release occurring on the Fe-protein drive the process. Hypothetically, P-clusters provide a reservoir of reducing equivalents that are transferred to substrate bound at FeMoco. Electrons are transferred one at a time from Fe-protein but the P-cluster and M center have electron buffering capacity, allowing successive two-electron transfers to, and protonations of, bound substrates.8 Neither component protein will reduce any substrate in the absence of its catalytic partner. Also, apoprotein (with any or all metal-sulfur clusters removed) will not reduce dinitrogen. 

The carbene-bound alkyl groups are acidic pX [(CO)5Cr=C(OMe)Me in H2O] 12.3 and can be easily deprotonated and alkylated [45,211,212] or acylated [213] (Figure 2.16). Stereoselective aldol-type additions can be realized with the aid of Fischer-type alkylcarbene complexes [214-216]. In these reactions the metallic fragment can either play the role of a bulky carbonyl group or stabilize a given conformation of the substrate by chelate formation [216,217]. 

NMR and kinetic studies have been conducted with the hope of providing more details about the position and conformation of the polypeptide substrate in cAMP-dependent protein kinase. These have served to narrow down the possible spatial relationships between enzyme bound ATP and the phosphorylated serine. Thus, a picture of the active site that is consistent with the available data can be drawn (12,13,66,67). Although these studies have been largely successful at eliminating some classes of secondary polypeptide structure such as oi-hellces, 6-sheets or an obligatory 6-turn conformation 66), the precise conformation of the substrate is still not known. The data are consistent with a preference for certain 6-turn structures directly Involving the phosphorylated serine residue. However, they are also consistent with a preference or requirement for either a coil structure or some nonspecific type of secondary structure. Models of the ternary active-site complexes based on both the coil and the, turn conformations of one alternate peptide substrate have" been constructed (12). These two models are consistent with the available kinetic and NMR data. 

Flaromy TP, Raleigh J, Sundaralingam M (1980) Enzyme-bound conformations of nucleotide substrates. X-ray structure and absolute configuration of 8,5 -cycloadenosine monohydrate. Biochemistry 19 1718-1722... 

Nonetheless, this raises the question of what is an appropriate definition of the NAC. Mulholland suggests a definition that is much less arbitrary the NAC is the conformation of the substrate bound to the enzyme, and what is critical then is the free energy needed to form this conformation in aqueous solution. MulhoUand estimates this energy as 4-5 kcal mol through a free energy perturbation calculation and MD using AMI and CHARMM. This is half the estimate of Bruice and suggests that formation of the NAC is only partly responsible for the catalytic effect afforded by CM. 

Chymotrypsin hydrolysis of spin-labeled ester substrates was studied by Electron Nuclear Double Resonance and molecular modeling methods (Wells et al., 1994). The spin-labeled acyl-enzyme was stabilized in low temperatures, and conformations of the substrate in the active site have been assigned from the experiments - both free in solution and in the active site. Conclusions from this study are that significant torsional alteration in the substrate s structrue occurs between its "free" form in solution and its bound form in the active site. The enzyme does not "recognize" the solution structure, but an altered one, that is steieospecifically complementary to the surface of the active site. 

Fig. 27 A representation of the conformation of the substrate analog GppNp bound to Ras. Dashed lines indicate hydrogen-bonding interactions. In this drawing the NH moiety that joins the (i- and y phosphoryl groups in the X-ray structure is replaced by oxygen, as in the natural substrate.

Conformation of Bound Inhibitor. Another area under investigation focuses on the conformations) of the substrate and an inhibitor (MTX) bound to DHFR. The form of the DHFR/MTX complex is known from the crystallographic studies of Kraut et al. (8-10). However, the orientation of the bound pterin ring in the reactive DHFR/DHF is known to differ dramatically from the MTX crystal structure (20). Basically, these differences arise because there are two possible orientations of the pterin ring in the active site one is flipped by 180° with respect to the other. Isotope labelling experiments on THF show that the reactive DHF must be bound in the conformation flipped from that observed by x-ray for MTX. In order to understand these differences, we ran simulation studies on altered forms of bound MTX and DHF to investigate the structural and energetic properties of these systems. 

P. aeruginosa AAC(3) for the NMR determination of bound substrate conformation," and the Streptomyces... 

Conversion of the C-terminal fragment to an activation-competent conformation is the first of several rearrangements of methionine synthase that we would like to study. Computations would complement the structure determinations, in the best Lipscomb tradition, by examining not only the static pictures of various conformers but also the likely pathways for interconversion of the structures (52) and the mechanisms for activation of bound substrates (55). 

An acid catalysis leads to protonation of the basic O atom of epoxide and favors the 5-exo-tet approach of the OH group with formation of TM 7.12. On the contrary, a specific antibody developed as a biocatalyst directs the recyclization of 3 by the disfavored 6-endo-tet route and exclusive formation of TM 7.11. This outcome is explained by the preferred conformation of 3 in the active site of the antibody, which closely resembles those on the 6-endo-tet route. Calculations revealed the energy of the transition state for the 5-exo-tet route to TM 7.12 1.8 kcal/mol is lower than for the 6-endo-tet route to TM 7.11. Selective lowering of the TS energy on the route to TM 7.11 is the result of preorientation of bound substrate 3 in the active site of the antibody, an important aspect of the mechanism of many enzymatic reactions. Remember that a less than 3 kcal/mol difference in the transition-state energy on the routes to enantiomeric products assures nearly 100 % e.e. (Sect. 3.4). Similarly, a small energy difference in activation energies determines the direction of chemoselective reactions. 

Strictly speaking, the conformations and relative geometries of the reactants must be known over the entire reaction coordinate moreover, there are indications that the transition states in enzyme reactions, which often have very different preferred conformations from those of the bound substrates, may be more tightly bound to the enzyme than either the starting materials or the products (1). 

In other cases, new asymmetric centers may be built into the substrate so that the stereochemical course of the overall reaction may be elucidated. The preferred conformation of the natural substrate when bound to the enzyme may be deduced and regions in the space around the enzyme-bound substrate where substituents can be tolerated may be inferred. 

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