The potency of thrombin inhibition by 4-methyl-1-[N2-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl)-sulfony l]- L-arginyl]-2-piperidinecarboxylic acid (MQPA) depended on the stereoconformation of the 2-piperidinecarboxylic acid moiety. Ki values for bovine alpha-thrombin were 0.019 microM with (2R,4R)-MQPA, 0.24 microM with (2R,4S)-MQPA, 1.9 microM with (2S,4R)-MQPA, and 280 microM with (2S,4S)-MQPA. (2R,4R)-MQPA of the four stereoisomers of MQPA was also the most potent inhibitor for other trypsin-like serine proteases with Ki values of 5.0 microM for trypsin, 210 microM for factor Xa, 800 microM for plasmin, and 1500 microM for plasma kallikrein. Examination of the potency of thrombin inhibition by arginine derivatives related to MQPA in structure suggested the presence of a specific binding site for the carboxamide portion (C-terminal side). The relative inhibitory potency of the four stereoisomers of MQPA for trypsin was nearly identical with that for thrombin, suggesting that the specific binding site for the carboxamide portion is present in both enzymes. Modification of thrombin by phosphopyridoxylation or the presence of heparin did not significantly alter the binding of MQPA.

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Holopropanediol dehydratase (EC 4.2.1.28) was resolved completely into the apoenzyme and corrinoid(s) upon gel filtration on a column of Sephadex G-25 in the absence of potassium ion. The apoprotein obtained by this resolution procedure could be reconstituted into the catalytically active holoenzyme by the incubation at 37' with added B12 coenzyme in the presence of potassium ion. The recovery of dehydratase activity was nearly quantitative. The major corrinoid recovered was identified as 5 '-deoxyadenosylcobalamin. Of inactive complexes between the apoenzyme and irreversible cobamide inhibitors of propanediol dehydratase, cyanocobalamin-and methylcobalamin-apoenzyme complexes were also mostly resolved upon the gel filtration in the absence of both potassium ion and the substrate, yielding the apoenzyme which was reconstitutable into the active holoenzyme. Hydroxocobalamin-apoenzyme complex, however, was hardly resolvable under the same conditions. In the presence of both potassium ion and the substrate, gel filtration of the holo-I t has been generally considered that vitamin B12 coenzyme or its analogs bind to the apoprotein of propanediol dehydratase (DL-1 ,Zpropanediol hydro-lyase, EC 4.2.1.28) or to those of many other Ble coenzyme-dependent enzymes almost irreversibly and the resolution of B12 coenzyme-apoenzyme complex (holoenzyme) or coenzyme analog-apoenzyme complex into the active apoprotein and corresponding cobalamin is usually accompanied by a significant loss of the enzyme activity. Charcoal treatment or exhaustive dialysis was found not to be effective. An acid ammonium sulfate resolution procedure, which was effectively used for mammalian methylmalonyl-CoA mutase (Lengyel et al., 1960) and ethanolamine deaminase (Kaplan and Stadtman, 1968b), led to a marked decrease in the propanediol dehydratase activity (T. Toraya, S. Shimizu, and S. Fukui, 1969, unpublished results).During the course of an investigation on factors necessary for binding of B12 coenzyme or its analogs to the apoprotein of propanediol dehydratase, our attention was drawn to the function of a monovalent cation which was required as a cofactor by this enzyme. Although it has previouslycbeen reported that propanediol dehydratase and many other coenzyme BIZ dependent enzymes show an absolute requirement for a monovalent cation, such as potassium ion, for their catalytic activitieslSmiley and Sobolov. Receiued March 15, 1971. A preliminary report of portions of this work has been published (Toraya er al., 1970).To whom correspondence should be addressed. enzyme or coenzyme analog-apoenzyme complexes resulted in negligible resolution. When gel filtration of the holoenzyme was carried out in the presence of potassium ion only, the holoenzyme was found to be resolved according to the kinetics of first-order reaction. These experiments indicate that potassium ion plays an essential role in the binding of the apoenzyme with coenzyme B I~ or its analogs except for hydroxocobalamin. Only hydroxocobalamin could bind to the apoenzyme even in t...

In the presence of dGTP, 5'-deoxyadenosylcobalamin (coenzyme B12) rapidly reacts with equimolar Lactobacillus leichmannii ribonucleotide reductase and excess dihydrolipoate. The spectral changes in the visible and ultraviolet closely correspond to those predicted for partial conversion to cob(II)alamin, but the deoxyadenosyl moiety cannot be trapped in a pool of 5'-deoxyadenosine. This distinguishes the reaction from irreversible degradation of coenzyme to cob(I1)alamin and 5'-deoxyadenosine which is lo5 times slower. The rapid reaction has kinetics approximating first order with a rate constant of about 38 sec-' at 37", and at equilibrium 20-40 of the coenzyme appears to be converted to the cob(I1)alamin-like species. The rapid reaction is completely reversed by a temperature drop from 37 to 5 " . Similar sensitivity to temperature has been demonstrated for rates of exchange of the 5' protons of the coenzyme with water, coenzyme degradation, and ribonucleotide reduction, but coenzyme degradation is not reversed by temperature decrease. Plots of the equilibrium position of the rapid reaction cs. temperature show a marked transition at 29" and a similar transition is observed for difference spectra of the enzyme compared at different temperatures. The implied conformation change of the enzyme at 29" probably involves movement of tryptophan residues to a less polar region or movement of charged residues in the vicinity of tryptophan. The equilibrium position for the rapid spectral change is less favorable when dATP, dCTP, or the arabino analog of ATP is substituted for P revious studies on the 5'-deoxyadenosylcobalamin-dependent reduction of ribonucleoside triphosphates to 2'deoxyribonucleoside triphosphates by dithiols in the presence of the reductase from Lactobacillus leichmannii have not succeeded in obtaining direct evidence for a reactive cobamide intermediate. Such an intermediate is nevertheless considered to be formed from the coenzyme on the basis of the following indirect evidence: (1) the hydrogens of the cobalt-bound methylene group in the coenzyme exchange with water in the presence of the enzyme, a dithiol, and an allosteric activator ; (2) the coenzyme is slowly degraded to cob(I1)alamin and 5 '-deoxyadenosine in the presence of

SummaryMCI–9042, (±)–1–[2–[2–(3–methoxyphenyl)ethyl]phenoxy]–3–(dimethylamino)–2–propyl hydrogen succinate hydrochloride inhibited platelet aggregation induced by collagen and secondary aggregation by ADP or epinephrine at 10−6 M level in platelets of various species. The antiplatelet effect of MCI–9042 was potentiated in aggregation induced by a combination of serotonin with collagen. IC50 value of human platelet aggregation by the serotonin plus collagen was 1.0 × 10−7 M. MCI–9042 inhibited serotonin release accompanied with collagen-induced platelet aggregation, while it did not affect serotonin uptake into platelet. MCI–9042 also potently inhibited the S2-serotonergic receptor-mediated contraction of rat caudal artery by serotonin in a competitive manner with a Ki value of 1.79 × 10–8 M, while Si receptor- or adrenergic receptor-mediated vasoconstriction was inhibited more weakly. Platelet adhesiveness, c-AMP level in platelets and the conversion of arachidonic acid to thromboxane A2 were not influenced by MCI–9042. These results suggest that MCI–9042 is a selective S2-serotonergic receptor antagonist, exhibiting the inhibition of S2-serotonergic potentiated platelet aggregation and the suppression of blood vessel constriction mediated by S2-serotonergic receptor.