Formation of a short (less than 2.5 angstroms), very strong, low-barrier hydrogen bond in the transition state, or in an enzyme-intermediate complex, can be an important contribution to enzymic catalysis. Formation of such a bond can supply 10 to 20 kilocalories per mole and thus facilitate difficult reactions such as enolization of carboxylate groups. Because low-barrier hydrogen bonds form only when the pKa's (negative logarithm of the acid constant) of the oxygens or nitrogens sharing the hydrogen are similar, a weak hydrogen bond in the enzyme-substrate complex in which the pKa's do not match can become a strong, low-barrier one if the pKa's become matched in the transition state or enzyme-intermediate complex. Several examples of enzymatic reactions that appear to use this principle are presented.

The proposal that low barrier (i.e. short, very strong) hydrogen bonds (LBHBs) 1 play a role in enzymatic catalysis was first put forth in 1993 and 1994 (1-4). The proposal was accepted by some but rejected by others (5-8). Initial rejection on theoretical grounds has been followed by increasing experimental support, and recent improvements in theory have been able to account for the experimental observations of LBHBs in enzymes (9 -15). In this minireview we will explain the original proposal, summarize the experimental data from the past few years, and argue that LBHBs do play important roles in enzymatic reactions. Properties of Hydrogen BondsThe strength of a hydrogen bond depends on its length and linearity, the nature of its microenvironment, and the degree to which the pK values of the conjugate acids of the heavy atoms sharing the proton are matched. In water, the hydrogen-bonded oxygens are separated by ϳ2.8 Å, and the ⌬H of formation is ϳ5 kcal mol Ϫ1 . The hydrogen bonds in water are, however, weak because of the poor pK match between the participating oxygen atoms. Because the pKs of H 3 O ϩ and H 2 O are Ϫ1.7 and 15.7, respectively, the proton in the structure H 2 O⅐⅐⅐H-OH is tightly associated with the OH Ϫ group as a water molecule. In the gas phase, where the dielectric constant is low, hydrogen bonds between heteroatoms with matched pKs can be very short and strong, and experimental as well as calculated values of ⌬H of formation can approach 25 or 30 kcal mol Ϫ1 (16, 17). Likewise, in crystals hydrogen bonds can be very strong. The O-O distance in the ion [H- O⅐⅐⅐H⅐⅐⅐O-H] -in a crystal of a chromium complex is only 2.29 Å (18,19). In organic solvents, strong hydrogen bonds can also form, although the ⌬H of formation probably never exceeds 20 kcal mol Ϫ1. Recent calculations suggest that once the dielectric constant is at least 6 the strength of a strong hydrogen bond levels off at a level about half that in the gas phase (13,17). Because the active site of an enzyme is no longer aqueous once it has closed around a substrate, the properties of hydrogen bonds in organic solvents are highly pertinent to enzymatic catalysis.What happens energetically as hydrogen bonds become shortened can be seen in Fig. 1. Structure A represents the situation in water, where the hydrogen is firmly attached to either the lefthand or right-hand oxygen and is more loosely bonded to the other one, with an O-O distance of Ն2.8 Å. There is an energy barrier between the two possible positions of the hydrogen, with the zero point energy levels shown in Fig. 1. Such a hydrogen bond is essentially electrostatic, and the covalent O-H bond is the usual 0.9 -1.0 Å in length.As the overall O-O distance is shortened, the energy barrier drops until it reaches the zero point energy level at an O-O distance of ϳ2.5 Å (Fig. 1B); this is a LBHB. The ⌬H of formation has increased to 15-20 kcal/mol, and the hydrogen can now move freely between the two oxygens. In crystals containing LBHBs, neutron diffraction shows the hydrogen dif...

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Synopsis Pyruvate carboxylase (PC) is a biotin-containing enzyme that catalyses the HCO3−- and MgATP-dependent carboxylation of pyruvate to form oxaloacetate. This is a very important anaplerotic reaction, replenishing oxaloacetate withdrawn from the Krebs cycle for various pivotal biochemical pathways. PC is therefore considered as an enzyme that is crucial for intermediary metabolism, controlling fuel partitioning toward gluconeogenesis, lipogenesis and insulin secretion. The enzyme was discovered in 1959 and over the last decade there has been much progress in understanding its structure and function. PC from most organisms is a tetrameric protein that is allosterically regulated by acetyl CoA and aspartate. High resolution crystal structures of the holoenzyme with various ligands bound have recently been determined, and have revealed details and the relative positions of the biotin carboxylase, carboxyltransferase and biotin carboxyl carrier domains, and also a unique allosteric effector domain. In the presence of the allosteric effector, acetyl CoA, the biotin moiety transfers the carboxyl group intermediate between the biotin carboxylase domain active site on one polypeptide chain and the carboxyltransferase active site on the adjacent antiparallel polypeptide chain. In addition, the bona fide role of PC in the non-gluconeogenic tissues has been studied using a combination of classical biochemistry and genetic approaches. The first cloning of the promoter of the PC gene in mammals and subsequent transcriptional studies reveal some key cognate transcription factors regulating tissue-specific expression. This review summarizes these advances and also offers some prospects in terms of future directions for the study of this important enzyme.

Biotin-dependent multifunctional enzymes carry out metabolically important carboxyl group transfer reactions and are potential targets for the treatment of obesity and type 2 diabetes. These enzymes use a tethered biotin cofactor to carry an activated carboxyl group between distantly spaced active sites. The mechanism of this transfer has remained poorly understood. Here we report the complete structure of pyruvate carboxylase at 2.0 angstroms resolution, which shows its domain arrangement. The structure, when combined with mutagenic analysis, shows that intermediate transfer occurs between active sites on separate polypeptide chains. In addition, domain rearrangements associated with activator binding decrease the distance between active-site pairs, providing a mechanism for allosteric activation. This description provides insight into the function of biotin-dependent enzymes and presents a new paradigm for multifunctional enzyme catalysis.

We have proposed previously that short, strong hydrogen bonds exist in enzyme active sites and that they are important in explaining enzymic rate enhancements. Here, we defend this proposal and provide evidence for likely changes of hydrogen bond strengths during enzymic catalysis.

Bacteria-mediated acquisition of atmospheric N2 serves as a critical source of nitrogen in terrestrial ecosystems. Here we reveal that symbiotic nitrogen fixation facilitates the cultivation of specialized fungal crops by leaf-cutter ants. By using acetylene reduction and stable isotope experiments, we demonstrated that N2 fixation occurred in the fungus gardens of eight leaf-cutter ant species and, further, that this fixed nitrogen was incorporated into ant biomass. Symbiotic N2-fixing bacteria were consistently isolated from the fungus gardens of 80 leaf-cutter ant colonies collected in Argentina, Costa Rica, and Panama. The discovery of N2 fixation within the leaf-cutter ant-microbe symbiosis reveals a previously unrecognized nitrogen source in neotropical ecosystems.

Stability constants for the Mg2+ and Cd2+ complexes of ATP, ADP, ATP alpha S, ATP beta S, and ADP alpha S have been determined at 30 degrees C and mu = 0.1 M by 31P NMR. Besides being of the utmost importance for determining species distributions for enzymatic studies, these constants allow an estimation of the preference of Cd2+ for sulfur vs. oxygen coordination in phosphorothioate complexes. Stability constants for Mg2+ complexes decreases when sulfur replaces oxygen (log K: ADP, 4.11; ADP alpha S, 3.66; ATP, 4.70; ATP alpha S, 4.47; ATP beta S, 4.04) because of (a) a statistical factor resulting from the loss of one potential phosphate oxygen ligand and (b) either an alteration in the charge distribution between oxygen and sulfur or destabilization of the chelate ring structure by loss of an internal hydrogen bond between an oxygen of coordinated phosphate and metal-bound water. Cd2+ complexes with sulfur-substituted nucleotides are more stable than those without sulfur (log K: ADP, 3.58; ADP alpha S, 4.95; ATP, 4.36; ATP alpha S, 4.42; ATP beta S, 5.44) because of the preferential binding of Cd2+ to sulfur rather than oxygen, which we estimate to be approximately 60 in CdADP alpha S and CdATP beta S. The proportion of tridentate coordination is estimated to be 50-60% in MgATP and MgATP beta S, approximately 27% in MgATP alpha S, approximately 16% in CdATP or CdATP beta S, but approximately 75% in CdATP alpha S. By analysis of the data of Jaffe and Cohn [Jaffe, E. K., & Cohn, M. (1979) J. Biol. Chem. 254, 10839], we conclude that the preference for oxygen over sulfur coordination to ATP beta S is 31 000 for Mg2+, 3100-3900 for Ca2+, and 158-193 for Mn2+. Proton NMR demonstrates that bidentate Cd2+ complexes form intramolecular chelates with the N-7 of adenine while Mg2+ nucleotides and the tridenate CdATP alpha S do not. An analysis of the 31P NMR line widths shows that the rate constants for dissociation of MgADP and MgATP are both 7000 s-1 while the association rate constants are 7 X 10(7) and 4 X 10(8) M-1 s-1, respectively. The observed dependence of the line width on nucleotide concentration is best explained by a base-stacking model at nucleotide concentrations above 5 mM.