Hexokinase isoenzymes

Many examples of product inhibition are to found. Some dehydrogenases are inhibited by NADH (a co-product of the reaction), e.g. PDH and isocitrate dehydrogenase (ICD), which are involved with the glycolysis and the TCA cycle are two such examples. Hexokinase isoenzymes in muscle (but not liver) and citrate synthase are inhibited by their products, glucose-6-phosphate and citrate respectively offering a very immediate fine tuning of reaction rate to match cellular requirements and possibly allowing their substrates to be used in alternative pathways. 

The substrate specificities of both mammalian and yeast hexo-kinases have been extensively studied (76,77). Nevertheless, work in this area continues both in the search for isoenzyme specific inhibitors and in increasingly detailed investigations of the catalytic mechanism. Recently potential transition state analogs PI-(adenosine-5 )-P3-glucose-6 triphosphate (Ap -glucose) and P1-(adenosine-5 )-P4-glucose-6 triphosphate (Ap.-giucose) were tested as inhibitors of four hexokinase isoenzymes. However, they were found to exhibit less affinity for the enzyme than either of the natural substrates alone (78). 

The uptake of glucose into the liver is independent of insulin, but liver has an isoenzyme of hexokinase (glucokinase) with a high K, so that as the concentration of glucose entering the liver increases, so does the... 

Brain hexokinase is inhibited by its product glucose-6-phosphate and to a lesser extent by adenosine diphosphate. The isoenzyme of hexokinase found in brain may be soluble in the cytosol or be attached firmly to mitochondria [2 and references therein]. An equilibrium exists between the soluble and the bound enzyme. The binding changes the kinetic properties of hexokinase and its inhibition by Glc-6-P resulting in a more active enzyme. The extent of binding is inversely related to the ATP ADP ratio, i.e. conditions in which energy utilization... 

A good example of allosteric inhibition is given by hexokinase (HK) isoenzymes of muscle. The product of the HK reaction, glucose-6-P allosterically inhibits the enzyme, so matching the phosphorylation of glucose to its overall metabolism, helps to regulate... 

Four tissue-specific isoenzymes of HK (denoted types I to IV) have been described type IV is usually known as GK and is found only in the liver. As noted previously, the hexokinases have a Km for glucose of about 1 mmol/1 whilst that for GK is about 10mmol/l. 

Even within a single organism, the same enzyme, as defined by the reaction it catalyses, can exist in more than one form, each of which is the prodnct of a different gene. These are known as isoenzymes. The properties of the two enzymes are nsnally different and this may explain the different roles of the isoenzymes within either a single type of tissue or within different tissnes, e.g. glncokinase and hexokinase in the hepatocyte (see below). 

Exploration of Bulk Tolerance at ATP Sites. Non-covalent type inhibitors have also been used to study bulk tolerance around the ATP binding sites. In this vein Hampton and co-workers have both synthesized and tested as inhibitors a large number of adenine nucleotide analogs (Figure 2f) to probe the bulk tolerance at a number of positions on the parent compound (28-31) These compounds have been used to study systematically the isoenzyme selectivity of adenylate kinases, hexokinases, thymidine kinases and pyruvate kinases with respect to bulk tolerance at many sites on the ATP molecule. Some of the most isoenzyme specific results were obtained with pyruvate kinase isoenzymes K,L and M using ADP derivatives. Here 3 -0Me-ADP was found to inhibit pyruvate kinase preferentially with a ratio of inhibitory potency of 7.6 6.0 1.0 for the K,M and L isoenzymes, respectively. Another compound, 8-NHEt-ADP, was selective for the M isoenzyme, giving a ratio of 7.1 1.2 1.0 for the M, K and L forms, respectively. 

The four forms of hexokinase found in mammalian tissues are but one example of a common biological situation the same reaction catalyzed by two or more different molecular forms of an enzyme. These multiple forms, called isozymes or isoenzymes, may occur in the same species, in the same tissue, or even in the same cell. The different forms of the enzyme generally differ in kinetic or regulatory properties, in the cofactor they use (NADH or NADPH for dehydrogenase isozymes, for example), or in their subcellular distribution (soluble or membrane-bound). Isozymes may have similar, but not identical, amino acid sequences, and in many cases they clearly share a common evolutionary origin. 

Hexokinase has a molecular weight of 102,000 and is composed of two identical subunits of 51,000 molecular weight each (20-22). In yeast, hexokinase exists as a mixture of two isoenzymes (23). These forms have been named A and B (23). These isoenzymes can be separated by chromatography and have been found to be chemically different (23, 24). Some of the earlier work with hexokinase was done with preparations that contained a mixture of isoenzymes. Work also was done with enzyme that was proteolytically modified. These variations undoubtedly have been responsible for some of the controversy concerning the properties of this enzyme. 

Four isoenzymes of hexokinases (types I-IV) constitute a family of enzymes that probably arose from a common ancestral gene by gene duplication and fusion events. Hexokinases I-III (M.W. 100,000) have a Km of about 0.1 mM and are allosterically inhibited by glucose-6-phosphate. Due to their low K, hexokinases I-III are saturated with the substrate glucose at... 

Hexokinases, the enzymes that catalyze the phosphorylation of glucose, are a family of tissue-specific isoenzymes that differ in their kinetic properties. The isoenzyme found in liver and p cells of the pancreas has a much higher than other hexokinases and is called glucokinase. In many cells, some of the hexokinase is bound to porins in the outer mitochondrial membrane (voltage-dependent anion channels see Chapter 21), which gives these enzymes first access to newly synthesized ATP as it exits the mitochondria. 

Fig. 22.12. Major sites of regulation in the glycolytic pathway. Hexokinase and phos-phofructokinase-1 are the major regulatory enzymes in skeletal muscle. The activity of pyruvate dehydrogenase in the mitochondrion determines whether pyruvate is converted to lactate or to acetyl Co A. The regulation shown for pyruvate kinase only occurs for the liver (L) isoenzyme.

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