Catalytic mechanism substrate binding

In order to establish the catalytic mechanism/substrate binding mode (B. Youn et al., manuscript in preparation), NtHCT was obtained in its apo form and also as a binary complex with coumaroyl CoA (9) and two different ternary complexes with -coumaroyl CoA(9)/shikimic acid (29) and -coumaroyl shikimate... 

Left side of Fig. 4 shows a ribbon model of the catalytic (C-) subunit of the mammalian cAMP-dependent protein kinase. This was the first protein kinase whose structure was determined [35]. Figure 4 includes also a ribbon model of the peptide substrate, and ATP (stick representation) with two manganese ions (CPK representation). All kinetic evidence is consistent with a preferred ordered mechanism of catalysis with ATP binding proceeding substrate binding. 

More recently, Kaiser and coworkers reported enantiomeric specificity in the reaction of cyclohexaamylose with 3-carboxy-2,2,5,5-tetramethyl-pyrrolidin-l-oxy m-nitrophenyl ester (1), a spin label useful for identifying enzyme-substrate interactions (Flohr et al., 1971). In this case, the catalytic mechanism is identical to the scheme derived for the reactions of the cycloamyloses with phenyl acetates. In fact, the covalent intermediate, an acyl-cyclohexaamylose, was isolated. Maximal rate constants for appearance of m-nitrophenol at pH 8.62 (fc2), rate constants for hydrolysis of the covalent intermediate (fc3), and substrate binding constants (Kd) for the two enantiomers are presented in Table VIII. Significantly, specificity appears in the rates of acylation (fc2) rather than in either the strength of binding or the rate of deacylation. 

Although these results have been interpreted in terms of a specific interaction between catalyst and substrate, the precise catalytic mechanism has not been firmly established. Indeed, the possibility that binding outside of the cavity of 16 leads to effective rate accelerations cannot be eliminated. Nevertheless, these preliminary results suggest that macrocyclic amines may surpass their progenitors, the cycloamyloses, in catalytic efficiency, and indicate a potentially fruitful direction for future research. 

Detailed three-dimensional models of P-gp-substrate complexes representing the various steps of the catalytic cycle would be significantly helpful in elucidating the molecular mechanism for substrate binding and release. The relatively poor 3D structural information available [14—16] and the complex mechanism for compounds undergoing P-gp-compound interactions explain why only a few groups have attempted to build and study 3D homology models for P-gp [56,58,60,70]. 

Fig. 20. Frame-by-frame series of stop-action pictures of the catalytic mechanism of RNase A at atomic resolution. Only the essential active site residues and the substrate (filled bonds) are shown. Frame 1, l e native enzyme. The sulfate ion which binds to the active site is shown. Frame 2, The Michaelis E-S complex with the dinucleotide CpA. The 2 oxygen which is deprotonated by His-12 is blackened. Frame 3, The transition state for...

Despite the large amount of biochemical and structural studies of sirtuins in complex with various substrates, cofactors and reaction products, the catalytic mechanism of this class of enzymes is still a matter of debate. SN -like [56] and SN -like [60] mechanisms have been inferred from structural studies but further biochemical and possibly structural studies will be required to clarify which mechanism is used by sirtuins. It should also be noted that another matter of debate concerns the mode of noncompetitive inhibition of sirtuins by the reaction product nicotinamide [62], various structural studies having highlighted different binding pockets for this molecule [63, 64]. 

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