**3. Structure of G6PD and enzymatic properties**

68 Dehydrogenases

*al.*, 1965).

random deactivation of X-chromosomes, about half of the cells will be normal and the other half will be deficient, although there is a wide range of variation around that average (Nance, 1964, Rinaldi*, et al.*, 1976). For this reason, total G6PD activity in heterozygous females can show variability between near-normal to near-deficient (Luzzatto & Battistuzzi, 1985, Segel, 2000). Deactivation of X-chromosome actualizes at random. Correspondingly, binomial distribution would be expected in deficiency level; the extent of this distribution depends on X-inactivation time in embryonic tissue and the number of cells in the embryo. Furthermore, random deactivation of one X-chromosome engenders genetic mosaics in heterozygous females (Luzzatto, 2006). As a result, G6PD mutations show the typical mendelian X-linked inheritance (Adam, 1961), severe G6PD deficiency is much more common in males than in females, and X-chromosome inactivation in heterozygous females for two different G6PD alleles indicate somatic cell mosaicism (Beutler*, et al.*, 1962, Gall*, et* 

The total length of the gene is about 18.5 kb on the X chromosome (xq28) and contains 13 exons. Exon 13 is about 800 nucleotides long and contains the translation stop codon (Nagel & Roth, 1989, Greene, 1993). The protein-coding region is divided into 12 segments, ranging in size from 12 to 236 bp (Martini*, et al.*, 1986). Exon and intron numbers and the exon sizes and sequences are conserved in higher eukaryotes (Nagel & Roth, 1989, Greene, 1993). The first exon contains no coding sequence and intron 2 between exons 2 and 3 is extraordinarily long, extending for 9,857 bp. The function of this long intron is unknown; it may be important for transcription or processing because compressed versions of the G6PD gene

The sequence of the whole G6PD gene is known (Chen*, et al.*, 1991). G6PD sequence analogy between humans and mice or rats is 87%. The analogy between the mouse and rat cDNA sequences is greater than humans with 93% similarity. Most of the sequence dissimilarity is in the 3´- UTR region, which has 600 nucleotides on average and contains a single polyA site (Nagel & Roth, 1989, Greene, 1993). G6PD gene promoter is embedded in a CpG island that starts about 680 nucleotides upstream of the transcription initiation site, extending about 1,050 nucleotides downstream of the initiation site, and ends at the start of the first intron (Chen*, et al.*, 1991). CpG island is conserved between some species (Martini*, et al.*, 1986, Toniolo*, et al.*, 1991), and has highly enriched guanine and cytosine residues, like characteristically in other housekeeping genes and this island appears to be preserved

The promoter of the G6PD gene contains a TATA-like, TTAAAT sequence, and a great number of stimulatory protein 1 (Sp1) elements (Philippe*, et al.*, 1994, Rank*, et al.*, 1994, Franze*, et al.*, 1998, Hodge*, et al.*, 1998). These Sp1-binding sites are essential for promoter activity (Philippe*, et al.*, 1994). Deletion analysis has uncovered that the "essential" segment

The transcribed region from the initiation site to the poly(A) addition site covers 15,860 bp. (Chen*, et al.*, 1991). The major 5'-end of mature G6PD mRNA in several cell lines is located

still have this largest intron in some species (Chen*, et al.*, 1991, Mason*, et al.*, 1995).

between humans and mice (Toniolo*, et al.*, 1991).

of the promoter is only about 150 bp (Ursini*, et al.*, 1990).

G6PD is a typical cytoplasmic, housekeeping enzyme and has been found in all cells from liver to kidney and organisms, from prokaryotes to yeasts, to protozoa, to plants and animals (Luzzatto & Battistuzzi, 1985, Antonenkov, 1989, Glader, 1999, Notaro*, et al.*, 2000). Inactive form of G6PD is a monomer with 515 amino acids and has a molecular weight of over 59 kDa (Rattazzi, 1968). The primary structure of the G6PD enzyme in humans has been determined from the sequence of full-length cDNA clones (Persico*, et al.*, 1986). Furthermore, the tertiary structure of the enzyme has been determined (Au*, et al.*, 1999). Dimer structure of the two subunits in the enzyme are symmetrically located across a complex interface of β-sheets (Au*, et al.*, 1999).

Activation of the enzyme requires NADP+ tightly binding to dimer or tetramer formation of enzyme. G6PD catalyses the first step of the oxidative pentose phosphate pathway and controls reaction velocity (Wrigley*, et al.*, 1972). In this first step, while G6PD catalyses the conversion of glucose 6-phosphate (G6P) to 6-phosphogluconolactone, at the same time it reduces NADP to NADPH (Au*, et al.*, 1999, Turner, 2000). Human G6PD has no activity with nicotinamide adenine dinucleotide (NAD) as coenzyme. Also, G6P is very specific for its substrate compared to other hexose phosphates (e.g., galactose 6-phosphate or mannose 6-phosphate) (Luzzatto & Battistuzzi, 1985, Glader, 1999). The G6P binding site is nearby lysine 205 in tertiary structure of the enzyme, and this amino acid has a critical role in electron transfer (Bautista*, et al.*, 1995). The NADP binding site is located nearby 38 to 43 amino acids; this region constitutes the Nterminus in tertiary structure encoded in exon 3. This site is important for stability of G6PD (Au*,* 

*et al.*, 1999). As an inhibitory effect, one of the products of G6PD reaction NADPH is an effective inhibitor (Luzzatto, 1967). Increase in NADP and decrease in NADPH as a result of whichever oxidative event in cells effect prepotently to increase G6PD activity (Luzzatto & Testa, 1978). Consequently, G6PD is the most important enzyme in biosynthesis reactions owing to enzyme property as NADPH reducer in its critical role in the cytoplasm (Koehler & Van Noorden, 2003).

Glucose-6-Phosphate Dehydrogenase

Deficiency and Malaria: A Method to Detect Primaquine-Induced Hemolysis *in vitro* 71

which NADP+ is reduced by glucose-6-phosphate with G6PD B+ as the catalyst is the standard for activity. Based on this, enzyme activity relative to G6PD B+ variants are classified as fast, normal, and slow in terms of electrophoretic mobility and as Classes I—V (Luzzatto, 1989, Beutler, 1990, Greene, 1993, Segel, 2000, Betke K, Brewer GJ, Kirkman HN, Luzzatto L,Motulsky AC, Ramot B, and Siniscalco M 1967). There are 5 classes for these variants. Class I includes chronic nonspherocytic hemolytic anemia with a severe enzyme deficiency (e.g., G6PD Minnesota, G6PD Tokyo, G6PD Campinas). Class II variants have severe enzyme deficiency without chronic nonspherocytic hemolytic anemia (e.g., G6PD mediterrian, G6PD Canton, G6PD Union, G6PD Kaiping). Class III variants includes medium or mild enzyme deficiency, with the activity at 10-60% of G6PD B+ (e.g., G6PD Aˉ). Class IV variants have a weak or no enzyme deficiency. The activity is 60-100 % of G6PD B+ (e.g., G6PD A+). Class V variants have increased enzyme activity (e.g., G6PD Hektoen)

**4.3.1.1 G6PD A+** is the most widely seen variant worldwide and also the first variant in which the nucleotide mutation and amino acid substitution were determined (Beutler, 1990). This Class IV variant has 90% of the enzyme activity of G6PD B+ (Luzzatto, 1989). This variant also called for the African variant cause widely seen in Africa; 20-40% of African men and 20% of African American men have this variant. It is faster than G6PD B+ electrophoretically and it does not cause hemolysis (Beutler, 1989). G6PD A+ derives from a single amino acid substitution of aspartic acid for asparagine at amino acid number 126, and

**4.3.1.2 G6PD Aˉ** is a Class II variant that has 10 to 20% of the activity of G6PD B+ and the same electrophoretically mobility as G6PD A+ (Luzzatto, 1989); 11% of African American men have this variant. Its half-life is 13 days. Three types of mutations have arisen with molecular studies. The most common mutation being at nucleotide number 202 is a result of a guanine to adenine mutation at amino acid number 68 substitution of valine to methionine (Beutler, 1989, Luzzatto, 1989, Beutler, 1990). The second one occurs at nucleotide number 680 as a result of a guanine to thymine mutation at amino acid number 227 substitution of arginine to leucine. And the third mutation occurs at the nucleotide number 968, as a result of a thymine to cytosine mutation at amino acid number 323 substitution of leucine to proline (Beutler, 1989). G6PD A + and G6PD Aˉ variants are defined as unique to Africa, but they can also be seen in Caucasian populations from Italy, Spain, Southeast Asia, Middle

**4.3.1.3 G6PD Mediterranean** is a widely seen variant in the Mediterranean region and Middle East. In addition, it is seen in the Indian subcontinent and other regions of the Americas (Beutler, 1991). This Class II variant has less than 10% of the enzyme activity of G6PD B+ and the electrophoretical mobility is similar with G6PD B+ (Luzzatto, 1989). Its halflife is only 8 days and DNA analysis identified two different point mutations. The first mutation is a result of a cytosine to thymine mutation at nucleotide number 563, at amino

this was the result of an adenine to guanine mutation at nucleotide number 376.

(Beutler, 1994, Segel, 2000).

*4.3.1. Some Important G6PD Variants* 

East and South America (Beutler, 1990).
