Substrate-binding

Peptide substrate then docJcs onto the protein kinase, in general presumably occupying a cleft along the C-terminal lobe, as exemplified by the peptide inhibitor in the PKA-AMPPNP-PKI and IRK-ATP structures. Catalysis appears to be via a dissociative transition state mecdianism and a planar phosphate intermediate [20, 22]. The incoming peptide hydroxyl is oriented via Asp-127, which is in turn further stabilized via a hydrogen bond to Asn-132. These latter two residues that 

Although it has not been established by X-ray crystallography that substrate binds to the copper by displacing the equatorial water, this is consistent with graphics modelling based on the 1.7 X-ray structure (Ito et al., 1994). This is consistent with the water proton relaxation data (Knowles et al., 1995) that show that a water proximal to copper can be titrated by the substrate dihydroxyacetone. 

The enzyme has an absolute requirement for a divalent cation, which could function in substrate binding, catalysis, or both. Radioactive Mn did not bind appreciably in the absence of substrate. With substrate or product present, 2 moles of metal were bound per subunit. Metal ions (Mn or Mg ) also enhanced the binding of substrates several-fold. Simultaneous binding of two unreactive fluorine analogs, one allylic the other homoallylic, did not enhance the binding of the metal ion. These experiments demonstrated that the metal ion is essential for catalysis and may not play an important role in substrate binding [6]. 

The earliest studies relative to the mechanism was the stereochemical work of Conforth, Popjak, and their collaborators [49]. They concluded that there was an inversion of configuration at C-1 of the allylic substrate, consistent with a concerted process, the new carbon-to-carbon bond being formed as the carbon-to-oxygen bond is cleaved. They also felt that elimination of a proton from C-2 of the isopentenyl moiety would not be concerted with this, since suprafacial (same side) reactions are generally considered unfavorable. To circumvent this, a 2-stage mechanism involving an electron donor X , with X being covalently linked to the initial condensation product, was proposed. The X residue is then lost simultaneously with elimination of the proton in an anti-mode (Fig. 12). 

Reed found that crystalline prenyltransferase could solvolyze the allylic substrates. This reaction required inorganic pyrophosphate and had a velocity of about 2% of the normal reaction rate [6]. Examination of the allylic product, either dimethylallyl alcohol or geraniol, revealed that C-1 had inverted and the csu-binol oxygen had come from water. Since the normal reaction involves inversion of C-1 and scission of C-O bond, the solvolysis seemed to be mimicking the normal reaction, with H2O replacing the organic portion of isopentenyl pyrophosphate in the catalytic site. This indicates that ionization of the allylic pyrophosphate is the first event, followed by condensation to form a new bond, then a hydrogen elimination from C-2 of the former isopentenyl moiety. Thus, there is an ionization-condensation-elimination sequence of events. 

Poulter and his group used another approach to demonstrate this mechanism [6]. 

Relative reactivities of allylic systems in nucleophilic substitution reactions 

Blevins and Tulinsky (1985) suggested two functions for the solvent at the chymotrypsin active site (1) solvation of the Asp—His—Ser catalytic triad, and (2) a guiding effect on the substrate in formation of the enzyme-substrate complex, provided by several waters at the end of the specificity site. X-Ray diffraction results have suggested a role of active-site water in determining the kinetics or equilibria of substrate binding for other proteins (Section IV). 

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Reversibly fonned micelles have long been of interest as models for enzymes, since tliey provide an amphipatliic environment attractive to many substrates. Substrate binding (non-covalent), saturation kinetics and competitive inliibition are kinetic factors common to botli enzyme reaction mechanism analysis and micellar binding kinetics. 

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. 

Although FeMo-cofactor is clearly knpHcated in substrate reduction cataly2ed by the Mo-nitrogenase, efforts to reduce substrates using the isolated FeMo-cofactor have been mosdy equivocal. Thus the FeMo-cofactor s polypeptide environment must play a critical role in substrate binding and reduction. Also, the different spectroscopic features of protein-bound vs isolated FeMo-cofactor clearly indicate a role for the polypeptide in electronically fine-tuning the substrate-reduction site. Site-directed amino acid substitution studies have been used to probe the possible effects of FeMo-cofactor s polypeptide environment on substrate reduction (163—169). Catalytic and spectroscopic consequences of such substitutions should provide information concerning the specific functions of individual amino acids located within the FeMo-cofactor environment (95,122,149). 

Ca.ta.lysis, Iridium compounds do not have industrial appHcations as catalysts. However, these compounds have been studied to model fundamental catalytic steps (174), such as substrate binding of unsaturated molecules and dioxygen oxidative addition of hydrogen, alkyl haHdes, and the carbon—hydrogen bond reductive elimination and important metal-centered transformations such as carbonylation, -elimination, CO reduction, and... 

P-Lactam antibiotics exert their antibacterial effects via acylation of a serine residue at the active site of the bacterial transpeptidases. Critical to this mechanism of action is a reactive P-lactam ring having a proximate anionic charge that is necessary for positioning the ring within the substrate binding cleft (24). 

The low detection limit, high sensitivity, and fast response times of chemoreceptor-based biosensors result primarily from the extremely high binding constants of the receptor R for the target substrate S. The receptor—substrate binding may be described... 

Receptor—substrate-binding constants are typically between 10 and 10, at equimolar ratios, implying that when a receptor—substrate... 

S Modi, MI Paine, MI Sutcliffe, L-Y Lian, WU Pnmi-ose, CR Wolfe, GCK Roberts. A model for human cytochrome P450 2d6 based on homology modeling and NMR studies of substrate binding. Biochemistry 35 4540-4550, 1996. 

Figure 4.8 The active site in all a/p barrels is in a pocket formed by the loop regions that connect the carboxy ends of the p strands with the adjacent a helices, as shown schematically in (a), where only two such loops are shown, (b) A view from the top of the barrel of the active site of the enzyme RuBisCo (ribulose bisphosphate carboxylase), which is involved in CO2 fixation in plants. A substrate analog (red) binds across the barrel with the two phosphate groups, PI and P2, on opposite sides of the pocket. A number of charged side chains (blue) from different loops as welt as a Mg ion (yellow) form the substrate-binding site and provide catalytic groups. The structure of this 500 kD enzyme was determined to 2.4 A resolution in the laboratory of Carl Branden, in Uppsala, Sweden. (Adapted from an original drawing provided by Bo Furugren.)...

The theory predicts that such proteins are built up of several subunits which are symmetrically arranged and that the two states differ by the arrangements of the subunits and the number of bonds between them. In one state the subunits are constrained by strong bonds that would resist the structural changes needed for substrate binding, and this state would consequently bind substrates weakly they called it the tense or T state. In the other state, called the R state, these constraints are relaxed. 

This concerted model assumes furthermore that the symmetry of the molecule is conserved so that the activity of all its subunits is either equally low or equally high, that is, all structural changes are concerted. Subsequently Daniel Koshland, University of California, Berkeley, postulated a sequential model in which each subunit is allowed independently to change its tertiary structure on substrate binding. In this model tertiary structural changes in the subunit with bound ligand alter the interactions of this... 

Zhu, X., et al. Structural analysis of substrate binding by the molecular chaperone DnaK. Science 272 1606-1614, 1996. 

Figure 11.6 A schematic view of the presumed binding mode of the tetrahedral transition state intermediate for the deacylation step. The four essential features of the serine proteinases are highlighted in yellow the catalytic triad, the oxyanion hole, the specificity pocket, and the unspecific main-chain substrate binding.

Inhibitors as well as substrates bind in this crevice between the domains. From the numerous studies of different inhibitors bound to serine pro-teinases we have chosen as an illustration the binding of a small peptide inhibitor, Ac-Pro-Ala-Pro-Tyr-COOH to a bacterial chymotrypsin (Figure 11.9). The enzyme-peptide complex was formed by adding a large excess of the substrate Ac-Pro-Ala-Pro-Tyr-CO-NHz to crystals of the enzyme. The enzyme molecules within the crystals catalyze cleavage of the terminal amide group to produce the products Ac-Pro-Ala-Pro-Tyr-COOH and NHs. The ammonium ions diffuse away, but the peptide product remains bound as an inhibitor to the active site of the enzyme. 

Figure 11.9 A diagram of the active site of chymotrypsin with a bound inhibitor, Ac-Pro-Ala-Pro-Tyr-COOH. The diagram illustrates how this inhibitor binds in relation to the catalytic triad, the strbstrate specificity pocket, the oxyanion hole and the nonspecific substrate binding region. The Inhibitor is ted. Hydrogen bonds between Inhibitor and enzyme are striped. (Adapted from M.N.G. James et al., /. Mol. Biol. 144 43-88, 1980.)...

Figure 11.10 Topological diagram of the two domains of chymotrypsin, illustrating that the essential active-site residues are part of the same two loop regions (3-4 and 5-6, red) of the two domains. These residues form the catalytic triad, the oxyanion hole (green), and the substrate binding regions (yellow and blue) including essential residues in the specificity pocket.

Krieger, M., Kay, L.M., Stroud, R.M. Structure and specific binding of trypsin comparison of inhibited derivatives and a model for substrate binding. /. Mol. Biol. 83 209-230, 1974. 

Active site Region of an enzyme where substrates bind. 

Inhibition The decrease of the rate of an enzyme-catalyzed reaction by a chemical compound including substrate analogues. Such inhibition may be competitive with the substrate (binding at die active site of die enzyme) or non-competitive (binding at an allosteric site). 

For a Michaelis-Menten reaction, ki = 7X10VAf- sec, /f i = 1 X lOVsec, and fe = 2 X lOVsec. What are the values of Ks and Does substrate binding approach equilibrium or does it behave more like a steady-state system ... 

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