Enzyme-bound conformation

Finally, the effect of neomycin-B flexibility on its enzymatic inactivation by Enterococcus faecalis APH(3 ) was tested. This enzyme catalyzes the transfer of a phosphate group from ATP to position 4 of ring I. In this case, the X-ray structure of the enzyme in complex with neomycin-B shows that the antibiotic structure recognized by the enzyme and the target RNA are remarkably similar (see Fig. 7) [40]. In fact, they mainly differ in the conformation adopted by the furanose ring of III (C3-endo and close to C2-endo for the RNA- and enzyme-bound conformations respectively). 

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... 

The process of constructing conformationally restricted analogs may fail for reasons other than steric hindrance or incorrect conformation. An enlightening example was encountered when the olefin isostere was used in place of the trans amide bond in the enzyme-bound conformation of CsA. As noted previously, the amide bond between positions 9,10 in CsA switches from cis in organic solution to trans in water and in the enzyme-bound conformation. When... 

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. ... 

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. 

Based on the three-dimensional structure of CHS, we proposed that the initiation/elongation/cyclization cavity serves as a structural template that selectively stabilizes a particular folded conformation of the linear tetraketide, allowing the Claisen condensation to proceed from C6 to Cl of the reaction intermediate.14 In contrast, CTAL formation can occur either in solution or alternatively while sequestered in the enzyme active site. In either case, enolization of the C5 ketone followed by nucleophilic attack on the Cl ketone with either a hydroxyl group (in solution) or the cysteine thiolate (enzyme bound) as the leaving group results in CTAL. Similar lactones are commonly formed as by-products of in vitro reactions in other PKS systems.36 38... 

Other enzyme-substrate or inhibitor interaction studies80 82 have been addressed, using a combination of STD and trNOE NMR experiments, in order to collect details on the substrate bound conformation (ligand perspective). In other cases, the availability of a labelled protein receptor83 have permitted to follow the induced chemical shift variations of the protein resonances upon ligand addition to the NMR tube by HSQC methods (protein perspective). 

If the crystal is destroyed on substrate addition and cross-linking is not possible, the only solution is to look for a different crystal form. It may be possible to crystallize the substrate-bound conformation of the enzyme by binding an inhibitor to the active site in solution, crystallizing the complex, and then diffusing the inhibitor out or exchanging it for substrate. All of these experiments are just searches for conditions under which there is no physical obstruction to the machinery of catalysis. 

Structural analysis of several non-NRPS adenyiation domains has provided significant insight into the basis for the multistep chemistry of NRPS A domains. Of note, the X-ray structures of 4-chlorobenzoate-CoA ligase bound to reaction intermediates showed two dramatically different orientations between the large and small domains. The enzyme bound to a substrate analogue was in a similar conformation as the described NRPS A-domain structures. In contrast, the structure of the enzyme bound to a product analogue revealed that... 

The different 3D-shapes adopted by aminoglycosides in the RNA- and enzyme-bound states suggest a possible structure-based chemical strategy to obtain antibiotics with better activity against resistant bacteria. Assiun-ing that, in these cases, some degree of conformational distortion of the substrates is required for enzymatic activity, it should be possible to design a conformationally locked oHgosaccharide that still retains antibiotic activity, but that is not susceptible to enzymatic inactivation (Fig. 8) [41]. 

Inhibitors of the angiotensin-converting enzyme (ACE) served as a test bed for the active-analog approach in which one tries to deduce the receptor-bound conformation... 

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. 

The pathway from enzyme-bound substrate to the transition state involves changes in the electronic configuration and geometry of the substrate. The enzyme itself is also not static. The ability to tightly bind the transition state requires flexibUity in the active site. Such flexibility has been experimentally demonstrated in many cases. A corollary to this is that the effectivity of enzyme catalysis can easily be influenced and regulated by conformational changes in the enzyme. An extensive consideration of the mechanisms of enzymes can be found in the works by J. Kraut (1988) and A. Fersht (1998). 

Enolase has a complex metal ion requirement,683 69 usually met by Mg2+ and Mn2+. From NMR studies of the relaxation of water protons, it was concluded that a Mn2+ ion coordinates two rapidly exchangeable water molecules in the free enzyme. When substrate binds, one of these water molecules may be immobilized and may participate in an addition reaction that forms phosphoenolpyruvate (reverse of reaction 13-15). A tightly bound "conformational" metal ion is located in the known three-dimensional structure in such a... 

---