Hexokinase substrate binding

Hexokinase provides an excellent example of induced fit as a means of using substrate binding energy. 

Hexokinase initially binds glucose, then the hex-okinase-glucose complex binds ATP. How do you explain the purpose of this substrate-binding pattern to a fellow student without using the induced-fit concept ... 

The induction of the correct geometry in the active site of an enzyme is paid for by a good substrate, with binding energy. An alternative explanation to that of induced fit is that some small molecules (e.g., HzO in the hexokinase example) bind nonproductively, i.e., their small size allows them to assume many orientations with respect to the other substrate (ATP in the case of hexokinase) that do not lead to reaction. Large substrates are restricted in motion and are held in a catalytically correct orientation millions of times more often during molecular vibrations than is, say, water. 

Hexokinase does not yield parallel reciprocal plots, so the Ping Pong mechanism can be discarded. However, initial velocity studies alone will noi discriminate between the rapid equilibrium random and steady-state ordered mechanisms. Both yield ihe same velocity equation and families of intersecting reciprocal plots. Other diagnostic procedures must be used (e.g., product inhibition, dead-end inhibition, equilibrium substrate binding, and isotope exchange studies). These procedures are described in detail in the author s Enzyme Kinetics behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems, Wiley-Interscience (1975),... 

The structure of hexokinase before binding its substrate is shown in red. The structure of hexokinase after binding its substrate is shown in green. 

The tertiary stmcture of a protein is the complete three-dimensional stmcture of the polypeptide chain. If multiple polypeptide chains are present, the arrangement of their polypeptide chains with respect to each other is the quaternary stmcture in such cases, enzymes are polymers conposed of two or more subunits (Table 3). In order to illustrate the tertiary stmcmre of a protein, the three-dimensional stmcture of the glycolytic enzyme hexokinase is shown in Fig. 3 with the aid of a space-filling model. Hexokinase catalyzes the phosphorylation of D-glucose with ATP. The groove in the middle of the stmcmre is where the substrates bind (Cantor Schimmel, 1980). 

The relatively unhindered domain motion in immunoglobulins may be contrasted with that observed in some enzymes, in which the domain motion occurs upon substrate binding during the catalytic cycle. The phenomenon has been established by crystal structure analysis of the different forms of yeast hexokinase [11], liver alcohol dehydrogenase [12] and citrate synthase [13], and appears to occur also in glyceraldehyde-3-phosphate-dehydrogenase. 

Figure 6.9 Models of hexokinase in space filling and wire frame formats, showing the cleft that contains the active site where substrate binding and reaction catalysis occur. At the bottom is an X-ray crystal structure of the enzyme active site, showing the positions of both glucose and ADP as well as a lysine amino acid that acts as a base to deprotonate glucose.

Hexokinase occurs in a wide variety of animals, plants and microorganisms. It is an example of the induced-fit model of substrate binding (S tion 5.2) in which glucose induces significant alterations in the tertiary structure of the enzyme. The positional changes of the amino acid residues within the... 

T"he extraordinary ability of an enzyme to catalyze only one particular reaction is a quality known as specificity (Chapter 14). Specificity means an enzyme acts only on a specific substance, its substrate, invariably transforming it into a specific product. That is, an enzyme binds only certain compounds, and then, only a specific reaction ensues. Some enzymes show absolute specificity, catalyzing the transformation of only one specific substrate to yield a unique product. Other enzymes carry out a particular reaction but act on a class of compounds. For example, hexokinase (ATP hexose-6-phosphotransferase) will carry out the ATP-dependent phosphorylation of a number of hexoses at the 6-posi-tion, including glucose. 

Computer models showing the shape of hexokinase (a) without and (b) with bound glucose. The enzyme folds around the substrate to bind it and isolate it from its aqueous environment. 

The x-ray structure for yeast hexokinase is also available. Thus, glucose analogs are now being used to elucidate minute details of the catalytic mechanism. Recently D- lose was used in crystallographic work to show that the 6-hydro ethyl group of the natural substrate is necessary for substrate-induced closure of the active-site cleft (81 ). This induced closure, which is observed with glucose binding (82), is believed to be a part of the induced fit mechanism postulated for hexokinase (83) ... 

We have seen how [S] affects the rate of a simple enzymatic reaction (S—>P) with only one substrate molecule. In most enzymatic reactions, however, two (and sometimes more) different substrate molecules bind to the enzyme and participate in the reaction. For example, in the reaction catalyzed by hexokinase, ATP and glucose are the substrate molecules, and ADP and glucose 6-phosphate are the products ... 

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