564 2. ANALOGS OF ENZYME REACTION COMPONENTS 



it can be termed a true pyridoxine analog; this substance will be discussed 

 separately at the end of this section. 



Effects on Pyridoxine Metabolisnn and Tissue Levels of Pyridoxine 



The only analog found in the early work to inhibit pyridoxal kinase strong- 

 ly is 2-ethyl-3-amino-4-ethoxymethyl-5-aminomethylpyridine (Hurwitz, 

 1952). This substance cannot be phosphorylated because of the lack of a 

 hydroxyl group at position 5, but has a rather high aJBinity for the yeast 

 enzyme {K^ = 0.073 mM) and competitively inhibits the phosphorylation 

 of pyridoxal. Certain analogs can be phosphorylated by this enzyme (e.g., 

 deoxypyridoxol, 3-deoxypyridoxol, and the 3-amino analog of pyridoxol) 

 (Umbreit and Waddell, 1949; Hurwitz, 1955 b), and presumably could re- 

 duce the phosphorylation of pyridoxal through substrate competition. Phos- 

 phorylation was shown to require a hydroxymethyl group at the 5-position 

 and the absence of a substituent at position 6. The more recent work of 

 McCormick and Snell (1961 ) demonstrated other potent inhibitors and made 

 it clear that the kinases from different sources vary markedly with respect 

 to affinity for the analogs (Table 2-35). Furthermore, the relative affinities 

 are fairly well correlated with the abilities to inhibit growth of the various 

 bacteria. The inhibitions are not always competitive and, in some instances, 

 increase with pyridoxal concentration. The 3-hydroxy group can be replaced 

 by an amino group or omitted without affecting affinity adversely, but 

 substitution in the 6-position reduces the affinity without necessarily abol- 

 ishing it. It is interesting that A^-methylpyridoxal is completely inactive as 

 an inhibitor. The most potent inhibitors of pyridoxal kinase are derivatives 

 obtained by the reaction of pyridoxal with various carbonyl agents. The 

 beef brain kinase is inhibited 50% by 0.00005 mM pyridoxal semicarbazone 

 and by 0.000065 mM pyridoxal azine (the product of the reaction of 2 

 pyridoxals with hydrazine), but the discussion of such inhibitions is more 

 pertinent to the subject of the carbonyl agents. 



Various enzymes oxidizing pyridoxol, pyridoxol-P, or pyridoxamine-P 

 have recently been found in liver, and are occasionally inhibited by analogs. 

 The oxidation of pyridoxol is competitively inhibited by deoxypyridoxol 

 (67% when pyridoxol is 10 mM and the analog is 12.5 mM) (Morino et al., 

 1960), while the oxidation of pyridoxol-P is competitively inhibited by 

 deoxypyridoxol-P {K„, = 0.02 mM, and K, = 0.35 mM) (Morisue et al, 

 1960). These enzymes thus follow the general rule that nonphosphorylated 

 analogs inhibit the reactions of nonphosphorylated substrates, and phos- 

 phorylated analogs inhibit the reactions of phosphorylated substrates. The 

 oxidative deamination of pyridoxamine-P is inhibited by pyridoxamine (the 

 latter is also deaminated at a slower rate) and rather weakly by pyridoxol 

 (Pogell, 1958). Wada and Snell (1961) examined the competitive inhibitions 

 of pyridoxol-P oxidase by a variety of substances (see accompanying tab- 



