9. SACCHARIDES: ALTERNATE ROUTES OF METABOLISM
439
ease of interconversion readily explains the presence of so many isomeric hexoses and pentoses throughout nature.
PPI and PKPE have been purified extensively. PPI has been obtained
from alfalfa (106) after a 350-fold purification that involved heating,
fractionation with (NH 4 ) 2 S0 4 , and adsorption and elution from Celite.
The enzyme appeared specific in its action on R-5-P and Ru-5-P. The
pH optimum occurred at 7.0, and the turnover number was 240,000
moles per minute per 100,000 gm. of enzyme. PKPE has been purified
700-fold from extracts of L. pentosus (101) by MnCl 2 and (NH 4 ) 2 S0 4
precipitation, adsorption and elution on calcium phosphate gel, and
heating. The K m for Xu-5-P was 5 X 10
4 M, whereas that for Ru-5-P
was somewhat greater (1χ10~
3 Μ).| As with PPI, the specificity of
PKPE appeared to be restricted to its normal substrates. Neither enzyme has been found to require a dissociable coenzyme (e.g., uridine
phosphate).
5. Transketolase
This enzyme has been purified from liver, spinach, and yeast (107,
108), the greatest purification effort being applied to yeast. As a result
of fractionation with acetone, alcohol, protamine, and ammonium Sulfate, a crystalline preparation has been obtained that is reportedly 1200
times as potent as the starting material and has a specific activity of
150 units per milligram (108).\ The best preparations from liver and
spinach have reported activities of 3.0 and 29 units per milligram,
respectively. No kinetic constants or other physical properties have been
reported for the enzyme; however, it is known that thiamine pyrophosphate and Mg
2+ are required for activity of transketolase. The equilibrium constant is about unity for the reaction. F-6-P -f- GLA-3-P -»
Xu-5-P + E-4-P (109).
The enzyme has broad specificity requirements, as shown in Table
III. The "donors" are similar (except hydroxypyruvate) in that their
first two carbon atoms resemble a glycolaldehyde unit, and the third
carbon (the first asymmetric carbon) possesses an L configuration. The
finding that Xu-5-P was active, not Ru-5-P, as a "donor" compound,
helped to make clear this configurational requirement. Acceptor aldehydes, on the other hand, show less specificity; most are phosphorylated,
but some, such as glycolaldehyde and glyceraldehyde, are not.
t Tabachnick et al. (104) have purified PPI 150-fold from spinach leaves and
PKPE 100-fold from rabbit muscle.
i One unit is the amount causing a change of 1.0 optical density at 340 τημ,
resulting from oxidation of added DPNH by glyceraldehyde-3-P0 4 (a product of the
reaction). Specific activity = units per minute per milligram protein.
439
ease of interconversion readily explains the presence of so many isomeric hexoses and pentoses throughout nature.
PPI and PKPE have been purified extensively. PPI has been obtained
from alfalfa (106) after a 350-fold purification that involved heating,
fractionation with (NH 4 ) 2 S0 4 , and adsorption and elution from Celite.
The enzyme appeared specific in its action on R-5-P and Ru-5-P. The
pH optimum occurred at 7.0, and the turnover number was 240,000
moles per minute per 100,000 gm. of enzyme. PKPE has been purified
700-fold from extracts of L. pentosus (101) by MnCl 2 and (NH 4 ) 2 S0 4
precipitation, adsorption and elution on calcium phosphate gel, and
heating. The K m for Xu-5-P was 5 X 10
4 M, whereas that for Ru-5-P
was somewhat greater (1χ10~
3 Μ).| As with PPI, the specificity of
PKPE appeared to be restricted to its normal substrates. Neither enzyme has been found to require a dissociable coenzyme (e.g., uridine
phosphate).
5. Transketolase
This enzyme has been purified from liver, spinach, and yeast (107,
108), the greatest purification effort being applied to yeast. As a result
of fractionation with acetone, alcohol, protamine, and ammonium Sulfate, a crystalline preparation has been obtained that is reportedly 1200
times as potent as the starting material and has a specific activity of
150 units per milligram (108).\ The best preparations from liver and
spinach have reported activities of 3.0 and 29 units per milligram,
respectively. No kinetic constants or other physical properties have been
reported for the enzyme; however, it is known that thiamine pyrophosphate and Mg
2+ are required for activity of transketolase. The equilibrium constant is about unity for the reaction. F-6-P -f- GLA-3-P -»
Xu-5-P + E-4-P (109).
The enzyme has broad specificity requirements, as shown in Table
III. The "donors" are similar (except hydroxypyruvate) in that their
first two carbon atoms resemble a glycolaldehyde unit, and the third
carbon (the first asymmetric carbon) possesses an L configuration. The
finding that Xu-5-P was active, not Ru-5-P, as a "donor" compound,
helped to make clear this configurational requirement. Acceptor aldehydes, on the other hand, show less specificity; most are phosphorylated,
but some, such as glycolaldehyde and glyceraldehyde, are not.
t Tabachnick et al. (104) have purified PPI 150-fold from spinach leaves and
PKPE 100-fold from rabbit muscle.
i One unit is the amount causing a change of 1.0 optical density at 340 τημ,
resulting from oxidation of added DPNH by glyceraldehyde-3-P0 4 (a product of the
reaction). Specific activity = units per minute per milligram protein.