464 2. ANALOGS OF ENZYME REACTION COMPONENTS 



Amylases 



Both a- and /^-amylases from barley are inhibited by heparin at concen- 

 trations around 0.1 mg/ml (Myrback and Persson, 1953 a, b). These inhi- 

 bitions show a striking pH dependence. At pH 5 or above there is no inhi- 

 bition whereas below pH 5 the inhibition increases rapidly and is very mark- 

 ed at pH 4.5. The isoelectric points of amylases usually lie between pH 5 

 and 6, so it is likely that the combination of heparin with the enzymes 

 below pH 5 is due to the positive charge arising in this range. The electro- 

 static nature of the binding is indicated by the protection afforded by high 

 concentrations of NaCl during the incubation of the enzyme and the heparin. 

 Once the inhibition is established, raising the NaCl concentration will not 

 reactivate. The presence of substrate also protects the enzyme, pointing to 

 a basically competitive mechanism. Human salivary amylase is also inhib- 

 ited by heparin but the critical pH is at least one unit higher, possibly 

 because of a higher isoelectric point than the barley enzymes (Astrup and 

 Thorsell, 1954). 



Enzymes Acting on Substrates Which Are Not Macroions 



Macroions interact with proteins generally when the conditions are favor- 

 able (e.g., when the total charges on protein and macroion are opposite, 

 although this is not a necessary condition, and when the ionic strength is 

 low), so it is not surprising that many enzymes whose substrates are small 

 molecules are inhibited. The combination is probably seldom at the active 

 center, but more often a bridging or covering of the active center by larger 

 molecules bound at many points and in no specific orientation. This type 

 of inhibition is, of course, independent of structural relations with the sub- 

 strate, but must always be considered in the use of macroionic inhibitors, 

 A few examples only will be mentioned. 



Prostatic acid phosphatase is potently inhibited by polyphloretin-phos- 

 phate in a noncompetitive fashion (Diczfalusy et al., 1953; Beling and Dicz- 

 falusy, 1959). K^ is given as 1.55 //g/ml. Polyestradiol phosphate is even 

 more inhibitory {K^ = 0.55 //g/ml). The inhibitions increase as the pH is 

 lowered below the pH^pt. Alginate of 556 residues and molecular weight 

 of 92,000 is also strongly inhibitory (^, = 0.0054 mM); the mechanism is 

 partially competition with substrate and partially interference with stabi- 

 lizing or protective substances present (Jeffree, 1957). The variation of the 

 inhibition with chain length is complex: chains of 10-100 residues inhibit 

 less, but below 10 the inhibition rises (Jeffree, 1956). A third potent inhi- 

 bitor is polyxenyl phosphate, a polydisperse, small molecular weight poly- 

 mer of branched chains and random coils, 66% inhibition being given 

 by 0.001 mM (Hummel et al., 1958). Polyhydroquinone is almost as potent, 

 polyethylenesulfonate and sulfonated polystyrene inhibit moderately, and 



