Enzyme molecular weight

Covalent modification of enzymes (molecular weight of several hundreds or thousands) by the incorporation of inorganic phosphate in the form of P03 (formula weight = 85), seems to represent a small chemical change in the enzyme yet is an important control mechanism of enzyme activity. Explain how phosphorylation can exert its controlling effect on the activity of the enzyme. 

Enzyme Molecular weight pi Collagenase activity M- min ) Metal content (moljmol protein ) ... 

Source of enzyme" Molecular weight Molecular activity1 Sedimentation coefficient (1013 X 20,w) rUcm "280 (nm/mg N) Electrophoretic mobility" (cm2/sec/VX 10-6) Ref. 

This is a small monomeric enzyme (molecular weight around 20000) that catalyses reduction of a double bond between carbon and nitrogen, converting 7,8-dihydrofolate into 5,6,7,8-tetrahydrofolate (Fig. 14). Hydride is transferred from the 4-pro-R position of NADPH to C-6, N-5 acquiring a proton [64-66]. 

Sedimentation, enzyme molecular-weight determination by, 287 Sedimentation coefiBcients of a-amylases, 309 of /3-amylases, 333 of phosphorylases, 348 Selenium, in hemiacetal rings of monosaccharides, 206 Seleno sugars, 232 Septanoses, 228... 

The enzyme contained three atoms of iron per molecule of the enzyme (molecular weight = approximately 140,000). All the iron in the native enzyme was shown to be in the divalent state, by ESR and colorimetric determinations. The enzyme was easily inactivated by an equimolar amount of H2O2 preincubated with the enzyme. Evidence indicates that inactivation is caused by simple oxidation of the ferrous ion to its ferric form (13). As Figure 1 shows, however, inactivation of the enzyme was completely counteracted by the presence of substrate, catechol. The reaction mixture contained 50 mM potassium phosphate buffer, pH 7.5, about 10 /xgrams of dialyzed enzyme, 5 /xM H2O2, and catechol (concentrations as indicated), in a final volume of 1.0 ml. All incubations were carried out at 24°C. for 10 minutes under anaerobic conditions. Activities were measured by the standard assay method (JO) with an aliquot of the mixture. 

The enzyme was found to contain about eight atoms of iron per molecule of enzyme (molecular weight 700,000), and the iron seemed to be in the trivalent state (3). The enzyme showed an absorption spectrum and ESR signal similar to those of pyrocatechase. The increase in the visible absorption and the disappearance of the ESR signal were also caused by adding substrate (protocatechuic acid). A similar spectral change was also observed when protocatechualdehyde, a competitive inhibitor, was added to the enzyme. Figure 2B shows the absorption spectra of native enzyme and its complex with substrate. 

The molar ratio of the arylazido-/S-alanine NAD and arylazido- 8-alanine to yeast alcohol dehydrogenase is based upon an enzyme molecular weight of 141,000 [Y. Hatefi, in Comprehensive Biochemistry (M. Florkin and E. Stolz, eds.), Vol. 14, p. 199. Elsevier, Amsterdam, 1966]. 

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