Glucose enzyme catalysis

Although most enzyme exchange studies have been investigated at equilibrium, the back exchange of labeled product while the reaction is proceeding in the forward direction can provide valuable information about enzymic catalysis. Under favorable conditions, one may utilize such isotope exchange data to learn about the order of product release and the presence of covalent enzyme-substrate compounds. One of the first systems to be characterized in this way was glucose-6-phos-phatase . ... 

Although precise positioning of the reactants is a fundamental aspect of enzyme catalysis, most enzymes undergo some change in their structure when they bind substrates. A particularly dramatic example is hexokinase, which catalyzes the transfer of a phosphate group from adenosine triphosphate (ATP) to glucose. 

At the present time, commercial isomerization processes based on enzymic catalysis are predominant, so only brief mention will be made of some of the nonenzymic processes that have been considered for commercialization in the past. Probably the major reasons for the current commercial use of enzymic rather than nonenzymic systems are that the nonenzymic systems so far developed result in products having one or more of the following defects too much ash, color, acid, off-flavor, a content of D-mannose or D-psicose, and high ratios of D-glucose to D-fructose. Probably, further advances in our understanding of the isomerization reaction and the mechanisms of the catalysis will lead to more efficient, nonenzymic processes that could replace the enzymic-isomerization systems now used commercially. 

The glycolysis of glucose proceeds through several steps involving enol intermediates to afford pyruvate, which can be converted into acetyl coenzyme A (acetyl-CoA) to participate in the Krebs cycle. Kinetic and crystal structure studies point to the key role played in enzyme catalysis by the stabilization of such intermediates on binding of enolate to the metal ion(s) of the enzymes. 

Preparation of the sample. Either semm or plasma (oxalated or heparinized) may be used for the determination. This may be freshly drawn blood from an animal. (Do not do this yourself your instructor will supply the sample.) See Chapter 1 for a discussion of the differences between semm, plasma, and whole blood. A 10- to 15-mL sample (20 to 30 mL whole blood) should be adequate for triplicate determinations by a class of 30 students. Fluoride should be added to prevent glycolysis, or breakdown of glucose, which can change the pH. The fluoride inhibits the enzyme catalysis causing glycolysis and stabilizes the pH for about 2 h. The tube used for collecting the sample can be rinsed with a solution of 100 mg sodium heparin plus 4 g sodium fluoride per 100 mL. The sample should be kept anaerobically, that is, stoppered to keep out atmospheric CO2. Since the analysis should be done on the day the blood is drawn, the solutions should be prepared ahead of time. 

Two distinguishing features of enzyme catalysis are noteworthy. There is usually an optimum pH value at which the enzyme activity is maximal. This is substantiated by many systems, for example, the enzyme-catalyzed oxidation of glucose (Figure 20.1). The second is the existence of an optimum temperature for maximum activity (see Johnson et al., 1954). 

Figure 13.1a depicts the classical double displacement mechanism proposed originally by Koshland as employed by retaining fi-exoglucosidases [3]. The enzyme active site contains two carboxylic acid (aspartate or glutamate) residues a general acid/base (carboxylic acid at the onset of enzyme catalysis) situated above the P-glucose substrate and a nucleophile (carboxylate) situated below. 

Enzymatic catalysis is also used in the synthesis of l-"C-labeled D-fructose and o-glucose firom mannitol and glucitol, respectively (Ogren and Langstrom 1998). The preparation of S-adenosyl-L-[ C]methionine (Guegen et al. 1982), [ C]epinephrine (Soussain et al. 1984), and [ C]daunorubicin (Eriks-Fluks et al. 1998) are other examples of enzyme catalysis. The nucleosides [ C]thymidine and [ C]-2 -arabino-2 -fluoro-P-5-methyl-uridine have been prepared by enzymes immobilized on hollow fiber membranes using [ C]formaldehyde as the labeled precursor (Hughes and Jay 1995). 

Yeast contains a number of enzymes, more particularly inyertase and zymase. Invertase catalyses the hydrolysis of sucrose to glucose and fructose (cf. the catalysis of this reaction by acids, p. 369). 

In 1878 the term enzyme, Greek for "in yeast," was proposed (8). It was reasoned that chemical compounds capable of catalysis, ie, ptyalin (amylase from sahva), pepsin, and others, should not be called ferments, as this term was already in use for yeast cells and other organisms. However, proof was not given for the actual existence of enzymes. EinaHy, in 1897, it was demonstrated that ceU-free yeast extract ("zymase") could convert glucose into ethanol and carbon dioxide in exactiy the same way as viable yeast cells. It took some time before these experiments and deductions were completely understood and accepted by the scientific community. 

Maltose phosphorylase proceeds via a single-displacement reaction that necessarily requires the formation of a ternary maltose E Pi (or glucose E glucose-l-phosphate) complex for any reaction to occur. Exchange reactions are a characteristic of enzymes that obey double-displacement mechanisms at some point in their catalysis. 

Figure 5.9 Models of hexo-kinase in space-filling and wireframe 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.

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