Paul Haake

Equilibrium constants for association of guanidinium and ammonium ions with oxyanions: The effect of changing basicity of the oxyanion

Abstract Using guanidinium and n-butylammonium cations (C+) as models for the positively charged side chains in arginine and lysine, we have determined the association constants with various oxyanions by potentiometric titration. For a dibasic acid, H2A, three association complexes may exist: K 1M = [CHA] [C + ] [HA − ] ; K 1D = [CA − ] [C + ] [A 2− ] ; K 2D = [C 2 A] [C + ] [CA − ] . For guanidinium ion and phosphate, K1M = 1.4, K1D = 2.6, and K2D = 5.1. The data for carboxylates indicate that the basicity of the oxyanion does not affect the association constant: acetate, pKa = 4.8, K1M = 0.37; formate, pKa = 3.8, K1M = 0.32; and chloroacetate, pKa = 2.9, K1M = 0.43, all with guanidinium ion. Association constants are also reported for carbonate, dimethylphosphinate, benzylphosphonate, and adenylate anions.

Catalysis by imidazolium ion and metal ions in the reaction of a phosphate diester

Abstract The rates of reaction of catechol cyclic phosphate in water and in acetonitrile-water demonstrate that imidazolium ion and metal ions (Na+, Mg2+, Zn2+) cause significant accelerations. These studies provide models for the potential role of cations in catalysis of reactions of phosphate anions by enzymes. In catalysis by Zn2+, we find that two to three imidazoles are required for coordination to Zn2+ for most effective catalysis. Enough water must be present to solvate imidazole and coordinate to Zn2+, indicating that a coordinated H2O is the nucleophile in Zn2+ catalysis. Product analysis also supports this conclusion.

Biological reactions at phosphorus: VII. The nature of metaphosphate ion, PO3−, as a reaction intermediate (1)

Abstract In order to improve our understanding of biological phosphorylations by “high-energy” compounds such as ATP, the hypothesis of metaphosphate ion as an intermediate in certain phosphorylation reactions has been critically examined. We have studied the rates and product composition for the solvolysis of the neutral form of N,N -dimethylphosphoroguanidinate (DMPG) at 30.5°C in various water-alcohol mixtures. The rates of solvolysis were found to decrease as the mole percent of the alcohol increased, but no systematic relationship with dielectric constant or Grunwald-Winstein y values was evident. A 1 : 1 correspondence between the percentage alkyl phosphate produced and the mole percent alcohol present was found with methanol, ethanol, and low concentrations of 2-propanol. At higher concentrations of 2-propanol, the product ratio favors water as nucleophile probably due to selective solvation of the metaphosphate precursor by water. These results indicate that metaphosphate mechanisms have a variable amount of nucleophilic participation. Although the reaction of phosphoroguanidines appears to involve metaphosphate ion as a free intermediate, analysis of results in the literature indicate that less reactive metaphosphate precursors react with nucleophilic participation. Extrapolation of these results to biological phosphorylations leads to the conclusion that nucleophilic participation may be an important feature of enzymic transition states due to the favorable orientation of nucleophile and incipient metaphosphate at enzymic active sites.

Regarding exchange of the 2-hydrogen of thiamine through the tetrahedral intermediate formed by nucleophilic addition

Abstract Kinetic data demonstrates that the exchange of hydrogen at the 2-position of thiazolium ions cannot occur through the tetrahedral intermediate formed by nueleophilic addition of hydroxide to the 2-position.

Distortion of OPO bond angles in phosphorus monoanions: Ab initio studies

Results are reported from ab initio studies of phosphinate anion H2PO2− and phosphate anion, (HO)2PO2− and the distortion of these anions by the biochemically important metal cations, Mg++ and Zn++. Ab initio results are reported from four basis sets for phosphinate ion and three basis sets for phosphate ion. We find that: 1) the results using the STO-3G∗ basis set are in reasonable agreement with experimental data and with the calculation done with the large 6−31+G∗ basis set and second-order Moller-Plesset perturbation theory; 2) that phosphinate anion is an appropriate analog for studying the interactions of phosphate monoanion; 3) that the interaction with Mg++ and Zn++ is enough to cause the deformation previously hypothesized; 4) that correlations we have noted from experimental results are supported by the computational data; 5) that the OPO angle contracts significantly more on metal interactions than we predicted from our previous experimental studies using the one-bond, PH coupling constant; and 6) density matrix elements for interaction of the 3s orbital at phosphorous and 1s orbital at hydrogen correlate with one-bond, PH coupling constants.

Phosphorus amides X. Rate acceleration due to solvation effects in the acid-catalyzed cleavage of phosphinamides. Application to acid-catalysis at enzymic active sites.

Abstract Phosphinamides undergo acid-catalyzed reaction with enhanced rates as the amount of water is decreased in acetonitrile from about 20 M to about 1 M. This phenomenon appears to be due to increased activity of acid and increased basicity of substrate as the concentration of water is decreased. The same effects may cause enhanced rates of acid-catalyzed reactions at enzymic active sites.

ROUND TABLE DISCUSSION ON CHEMISTRY AND MECHANISM

F. JORDAN (Rutgers University, Newark, N.J.): It is my pleasure to convene this brief round table discussion. Let me ask the panel’s opinion as to how the ylid is formed, since the pK, seems to be fairly high. Is ylid formation really rate-limiting? R. HOPMANN (University of Basel. Basel. Switz.): It’s correct that ylid formation is one of the most important steps in the enzymatic catalysis. Perhaps it’s possible to have the pyruvate carboxylate group remove the proton. Then you can have a concerted mechanism to produce the hydroxyethyl thiamin. P. HAAKE (Wesleyan University, Middletown, Conn.): Of course the pK is measured in water and the collapse of charge could make a significant perturbation so it may not be so difficult to generate that anion in the active site. It may not be as big a problem because it’s a positively charged acid going to a neutral base. HOPMANN: I assume you are referring to the measurements we have published in Nature. We tried to figure out the pK in aqueous solution but we also did some measurements in methanol. The pK of compounds like hydroxy acids or nitrogenous bases are shifted to higher pK values in alcohol as compared to water. But the pK of thiamin deprotonation to the ylid remains almost the same. A. SCHELLENBERGER (University of Halle. HalleWittenberg, G.D.R.): It is necessary to activate the enzymes prior to catalytic reactions. The substrate, as well as related compounds such as amides which cannot decarboxylate, can activate this enzyme. But if you carry out the normal reaction in the presence of amide you get a normal hyperbolic progress curve. That means that the amide or the substrate in some way must be concerned with the mechanism. R. KLUGER (University of Toronto, Toronto, Canada): Dr. Jordan asked whether ylid formation is going to be rate-limiting. In my talk I presented results that suggested that the nonenzymatic rate constant for addition of thiamin to pyruvate, incorporating the entropy effect of putting the two next to each other because they are on the enzyme, is almost sufficient to account for the entire rate factor on the enzyme. So that the enzyme appears not to have to do very much to bring those two together. My suspicion from our data is that the decarboxylation is the problem. The formation of lactyl-TTP will occur on the enzyme just by virtue of the fact that pyruvate and thiamin-PP are near one another. As for the assistance of a base to pull off a proton, from considering the pK that Dr. Hopmann has measured, we don’t have to propose any special mechanism for formation. What we do have to account for is how you accelerate the unimolecular decarboxylation of this adduct. Some proposals were made and I think that if I were looking in the future to understand this particular kind of enzyme, pyruvate decarboxylase, I would be looking for what happens after the intermediate forms, rather than how the intermediate forms. Again, we can speculate, but 1 think we should see what is going on in the enzyme during this unimolecular step. 1 I7