The specificity of hog liver folylpolyglutamate synthetase for folate substrates and for nucleotide and glutamate substrates and analogues has been investigated. The kinetic mechanism, determined by using aminopterin as the folate substrate, is ordered Ter-Ter with MgATP binding first, folate second, and glutamate last. This mechanism precludes the sequential addition of glutamate moieties to enzyme-bound folate. Folate, dihydrofolate, and tetrahydrofolate possess the optimal configurations for catalysis (kcat = 2.5 s-1) while 5- and 10-position substitutions of the folate molecule impair catalysis. kcat values decrease with increasing glutamate chain length, and the rate of decrease varies depending on the state of reduction and substitution of the folate molecule. Folate binding, as assessed by on rates, is slow. Dihydrofolate exhibits the fastest rate, and the rates are slightly reduced for tetrahydrofolate and 10-formyltetrahydrofolate and greatly reduced for 5-methyltetrahydrofolate and folic acid. The on rates for most pteroyldiglutamates are similar to the rates for their respective monoglutamate derivatives, but further extension of the glutamate chain results in a progressive decrease in on rates. Tetrahydrofolate polyglutamates are the only long glutamate chain length folates with detectable substrate activity. The specificity of the L-glutamate binding site is very narrow. L-Homocysteate and 4-threo-fluoroglutamate are alternate substrates and act as chain termination inhibitors in that their addition to the folate molecule prevents or severely retards the further addition of glutamate moieties. The Km for glutamate is dependent on the folate substrate used. MgATP is the preferred nucleotide substrate, and beta,gamma-methylene-ATP, beta,gamma-imido-ATP, adenosine 5'-O-(3-thiotriphosphate), P1,P5-di(adenosine-5') pentaphosphate, and free ATP4- are potent inhibitors of the reaction.

The ability of a commercial immobilized lipase preparation (Lipozyme) to hydrolyze the fatty acyl ester bonds of soybean phosphatidylcholine in organic media was investigated. Response surface methodology, based on a Modified Central Composite design, was employed to examine the effects on hydrolysis of solvent polarity, water, pH, duration and temperature of incubation, and the amounts of substrate and catalyst. A second‐order regression model was developed, which allows evaluation of the effects of these parameters, alone or in combination. Hydrolysis increased in a relatively straightforward manner in response to increases in incubation time and the amount of catalyst and was approximately constant over the range of substrate amounts studied. Solvent polarity had a profound effect on the degree of hydrolysis, and the qualitative and quantitative degrees of this effect were dependent upon the values of the other parameters studied. Conditions were identified where enzyme activity was strong in either nonpolar or polar solvents, with activity increasing as the polarity of the medium increased. Enzyme activity was minimum at about 37°C, increasing below and above this temperature. Activity was not affected by the presence of acid or base in the reactions. Increasing amounts of water stimulated enzyme activity in solvents more polar than hexane, while in less polar solvents water inhibited activity.

The complete purification and characterization of an extracellular lipase (acylglycerol acylhydrolase, EC 3.1.1.3) from K de/emar is describeeL The l'mal product was homo geneous as judged by electrophoresis in denaturing polyacrylnmide gels and by isoelectric focusing, and was shown by means of an activity stain to be lipolytic~ The purified enzyme had a monomer molecular weight of 30,300, an isoelectric point of 8.6, and approximately one monosa~ charide moiety per molecule. NYPermlnal sequence data (28 residues) and the amino acid composition of the lipase indicated that it corresponds to the product of a lipase-encoding cDNA previously isolated from/~ de/emar. Optimal activity occurred between pH 8.0 and 8.5. The activity and stability of the enzyme were maximum at 30~ Divalent cations were required for activity, with barium, calcium and manganese conferring mAYimum activity. Activation

Folylpolyglutamate synthetase was purified 30,000-150,000-fold from hog liver. Purification required the use of protease inhibitors, and the protein was purified to homogeneity in two forms. Both forms of the enzyme were monomers of Mr 62,000 and had similar specific activities. The specific activity of the homogeneous protein was over 2000-fold higher than reported for partially purified folylpolyglutamate synthetases from other mammalian sources. Enzyme activity was absolutely dependent on the presence of a reducing agent and a monovalent cation, of which K+ was most effective. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to tetrahydrofolate with the concomitant stoichiometric formation of MgADP and phosphate. Under conditions that resembled the expected substrate and enzyme concentrations in hog liver, tetrahydrofolate was metabolized to long glutamate chain length derivatives with the hexaglutamate, the major in vivo folate derivative, predominating. Enzyme activity was maximal at about pH 9.5. The high-pH optimum was primarily due to an increase in the Km value for the L-glutamate substrate at lower pH values, and the reaction proceeded effectively at physiological pH provided high levels of glutamate were supplied.

The abilities of four commercially available lipolytic enzymes [three immobilized lipases—Lipozyme IM‐20, SP‐435 (Novo Biolabs, Danbury, CT), and AY‐30/Celite (Amano Enzyme Co., Ltd., Troy, VA)—and a nonimmobilized Amano phospholipase B preparation] to hydrolyze mixtures of triacylglycerols (TG) and phospholipids (PL) were determined. All of the lipases hydrolyzed both types of substrates in water, with maximum rates of TG hydrolysis exceeding those of PL hydrolysis by between 20‐ and 200‐fold. The phospholipase B preparation was inactive against both TG and PL in water. All the enzymes showed some activity against lipids in hexane. The amount of activity was sharply dependent on the amount of water added to the reaction. Lipozyme IM‐20 and AY‐30/Celite hydrolyzed both TG and PL in hexane. Their estimated initial activities were between 10‐ and 100‐fold lower than those in water. Complete hydrolysis of the TG (measured as the hydrolysis of at least one ester bond in each molecule) was achieved, whereas only 40–60% of the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were hydrolyzed. Lipase SP‐435 was inactive against TG in hexane but hydrolyzed PC at a rate comparable to that seen in water, and it achieved complete hydrolysis of this substrate. Amano phospholipase B was inactive against TG in hexane but completely hydrolyzed the PC. The abilities of the enzymes to hydrolyze the TG, PC, and PE components of soybean soapstock, a by‐product of edible oil production, were also examined. Lipozyme IM‐20 hydrolyzed all the TG and a fraction of the PL in soapstock. SP‐435 and AY‐30/Celite were active only on soapstock that had been acidified prior to being dissolved in hexane. SP‐435 displayed significant activity only toward PE under these conditions, whereas AY‐30/Celite was active only toward TG. Phospholipase B was inactive against soapstock in hexane. The identity of the acid used in the acidification of soapstock affected the degree of hydrolysis by AY‐30/Celite, with nitric and hydrochloric acids giving the best activity.