Biochemical basis of glucokinase activation and the regulation by glucokinase regulatory protein in naturally occurring mutations.

Glucokinase (GK) has several known polymorphic activating mutations that increase the enzyme activity by enhancing glucose binding affinity and/or by alleviating the inhibition of glucokinase regulatory protein (GKRP), a key regulator of GK activity in the liver. Kinetic studies were undertaken to better understand the effect of these mutations on the enzyme mechanism of GK activation and GKRP regulation and to relate the enzyme properties to the associated clinical phenotype of hypoglycemia. Similar to wild type GK, the transient kinetics of glucose binding for activating mutations follows a general two-step mechanism, the formation of an enzyme-glucose complex followed by an enzyme conformational change. However, the kinetics for each step differed from wild type GK and could be grouped into specific types of kinetic changes. Mutations T65I, Y214C, and A456V accelerate glucose binding to the apoenzyme form, whereas W99R, Y214C, and V455M facilitate enzyme isomerization to the active form. Mutations that significantly enhance the glucose binding to the apoenzyme also disrupt the protein-protein interaction with GKRP to a large extent, suggesting these mutations may adopt a more compact conformation in the apoenzyme favorable for glucose binding. Y214C is the most active mutation (11-fold increase in k(cat)/K(0.5)(h)) and exhibits the most severe clinical effects of hypoglycemia. In contrast, moderate activating mutation A456V nearly abolishes the GKRP inhibition (76-fold increase in K(i)) but causes only mild hypoglycemia. This suggests that the alteration in GK enzyme activity may have a more profound biological impact than the alleviation of GKRP inhibition.

Glucose-induced conformational changes in glucokinase mediate allosteric regulation: transient kinetic analysis.

The transient kinetics of glucose binding to glucokinase (GK) was studied using stopped-flow fluorescence spectrophotometry to investigate the underlying mechanism of positive cooperativity of monomeric GK with glucose. Glucose binding to GK was shown to display biphasic kinetics that fit best to a reversible two-step mechanism. GK initially binds glucose to form a transient intermediate, namely, E* x glucose, followed by a conformational change to a catalytically competent E x glucose complex. The microscopic rate constants for each step were determined as follows: on rate k1 of 557 M(-1) s(-1) and off rate k(-1) of 8.1 s(-1) for E* x glucose formation, and forward rate k2 of 0.45 s(-1) and reverse rate k(-2) of 0.28 s(-1) for the conformational change from E* x glucose to E x glucose. These results suggest that the enzyme conformational change induced by glucose binding is a reversible, slow event that occurs outside the catalytic cycle (kcat = 38 s(-1)). This slow transition between the two enzyme conformations modulated by glucose likely forms the kinetic foundation for the allosteric regulation. Furthermore, the kinetics of the enzyme conformational change was altered in favor of E x glucose formation in D2O, accompanied by a decrease in cooperativity with glucose (Hill slope of 1.3 in D2O vs 1.7 in H2O). The deuterium solvent isotope effects confirm the role of the conformational change in the magnitude of glucose cooperativity. Similar studies were conducted with GK activating mutation Y214C at the allosteric activator site that is likely involved in the protein domain rearrangement associated with glucose binding. The mutation enhanced equilibrium glucose binding by a combination of effects on both the formation of E* x glucose and an enzyme conformational change to E x glucose. Kinetic simulation by KINSIM supports the conclusion that the kinetic cooperativity of GK arises from slow glucose-induced conformational changes in GK.

Dissection of the physiological interconversion of 5alpha-DHT and 3alpha-diol by rat 3alpha-HSD via transient kinetics shows that the chemical step is rate-determining: effect of mutating cofactor and substrate-binding pocket residues on catalysis.

3Alpha-hydroxysteroid dehydrogenases (3alpha-HSDs) catalyze the interconversion between 5alpha-dihydrotestosterone (5alpha-DHT), the most potent androgen, and 3alpha-androstanediol (3alpha-diol), a weak androgen metabolite. To identify the rate-determining step in this physiologically important reaction, rat liver 3alpha-HSD (AKR1C9) was used as the protein model for the human homologues in fluorescence stopped-flow transient kinetic and kinetic isotope effect studies. Using single and multiple turnover experiments to monitor the NADPH-dependent reduction of 5alpha-DHT, it was found that k(lim) and k(max) values were identical to k(cat), indicating that chemistry is rate-limiting overall. Kinetic isotope effect measurements, which gave (D)k(cat) = 2.4 and (D)2(O)k(cat) = 3.0 at pL 6.0, suggest that the slow chemical transformation is significantly rate-limiting. When the NADP(+)-dependent oxidation of 3alpha-diol was monitored, single and multiple turnover experiments showed a k(lim) and burst kinetics consistent with product release as being rate-limiting overall. When NAD(+) was substituted for NADP(+), burst phase kinetics was eliminated, and k(max) was identical to k(cat). Thus with the physiologically relevant substrates 5alpha-DHT plus NADPH and 3alpha-diol plus NAD(+), the slowest event is chemistry. R276 forms a salt-linkage with the phosphate of 2'-AMP, and when it is mutated, tight binding of NAD(P)H is no longer observed [Ratnam, K., et al. (1999) Biochemistry 38, 7856-7864]. The R276M mutant also eliminated the burst phase kinetics observed for the NADP(+)-dependent oxidation of 3alpha-diol. The data with the R276M mutant confirms that the release of the NADPH product is the slow event; and in its absence, chemistry becomes rate-limiting. W227 is a critical hydrophobic residue at the steroid binding site, and when it is mutated to alanine, k(cat)/K(m) for oxidation is significantly depressed. Burst phase kinetics for the NADP(+)-dependent turnover of 3alpha-diol by W227A was also abolished. In the W227A mutant, the slow release of NADPH is no longer observed since the chemical transformation is now even slower. Thus, residues in the cofactor and steroid-binding site can alter the rate-determining step in the NADP(+)-dependent oxidation of 3alpha-diol to make chemistry rate-limiting overall.

Alanine scanning mutagenesis of the testosterone binding site of rat 3 alpha-hydroxysteroid dehydrogenase demonstrates contact residues influence the rate-determining step.

Aldo-keto reductase (AKR1C) isoforms can regulate ligand access to nuclear receptors by acting as hydroxysteroid dehydrogenases. The principles that govern steroid hormone binding and steroid turnover by these enzymes were analyzed using rat 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD, AKR1C9) as the protein model. Systematic alanine scanning mutagenesis was performed on the substrate-binding pocket as defined by the crystal structure of the 3alpha-HSD.NADP(+).testosterone ternary complex. T24, L54, F118, F129, T226, W227, N306, and Y310 were individually mutated to alanine, while catalytic residues Y55 and H117 were unaltered. The effects of these mutations on the ordered bi-bi mechanism were examined. No mutations changed the affinity for NADPH by more than 2-3-fold. Fluorescence titrations of the energy transfer band of the E.NADPH complex with competitive inhibitors testosterone and progesterone showed that the largest effect was a 23-fold decrease in the affinity for progesterone in the W227A mutant. By contrast, changes in the K(d) for testosterone were negligible. Examination of the k(cat)/K(m) data for these mutants indicated that, irrespective of steroid substrate, the bimolecular rate constant was more adversely affected when alanine replaced an aromatic hydrophobic residue. By far, the greatest effects were on k(cat) (decreases of more than 2 log units), suggesting that the rate-determining step was either altered or slowed significantly. Single- and multiple-turnover experiments for androsterone oxidation showed that while the wild-type enzyme demonstrated a k(lim) and burst kinetics consistent with slow product release, the W227A and F118A mutants eliminated this kinetic profile. Instead, single- and multiple-turnover experiments gave k(lim) and k(max) values identical with k(cat) values, respectively, indicating that chemistry was now rate-limiting overall. Thus, conserved residues within the steroid-binding pocket affect k(cat) more than K(d) by influencing the rate-determining step of steroid oxidation. These findings support the concept of enzyme catalysis in which the correct positioning of reactants is essential; otherwise, k(cat) will be limited by the chemical event.

Structure-function relationships in 3alpha-hydroxysteroid dehydrogenases: a comparison of the rat and human isoforms.

3alpha-Hydroxysteroid dehydrogenases (3alpha-HSDs) inactivate steroid hormones in the liver, regulate 5alpha-dihydrotestosterone (5alpha-DHT) levels in the prostate, and form the neurosteroid, allopregnanolone in the CNS. Four human 3alpha-HSD isoforms exist and correspond to AKR1C1-AKR1C4 of the aldo-keto reductase (AKR) superfamily. Unlike the related rat 3alpha-HSD (AKR1C9) which is positional and stereospecific, the human enzymes display varying ratios of 3-, 17-, and 20-ketosteroid reductase activity as well as 3alpha-, 17beta-, and 20alpha-hydroxysteroid oxidase activity. Their k(cat) values are 50-100-fold lower than that observed for AKR1C9. Based on their product profiles and discrete tissue localization, the human enzymes may regulate the levels of active androgens, estrogens, and progestins in target tissues. The X-ray crystal structures of AKR1C9 and AKR1C2 (human type 3 3alpha-HSD, bile acid binding protein and peripheral 3alpha-HSD) reveal that the AKR1C2 structure can bind steroids backwards (D-ring in the A-ring position) and upside down (beta-face inverted) relative to the position of a 3-ketosteroid in AKR1C9 and this may account for its functional plasticity. Stopped-flow studies on both enzymes indicate that the conformational changes associated with binding cofactor (the first ligand) are slow; they are similar in both enzymes but are not rate-determining. Instead the low k(cat) seen in AKR1C2 (50-fold less than AKR1C9) may be due to substrate "wobble" at the plastic active site.

Steroid-binding site residues dictate optimal substrate positioning in rat 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD or AKR1C9).

Rat liver 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD or AKR1C9), a member of the aldo-keto reductase (AKR) superfamily, plays a pivotal role in the inactivation of circulating steroid hormones. It is the most thoroughly characterized HSD of the AKR superfamily and can be used as a template for structure-function studies in other AKR members such as rodent and human 3alpha-, 17beta- and 20alpha-HSDs. Based on the crystal structure of the E.NADP(+) testosterone ternary complex, there are ten residues that line the testosterone binding cavity: T24, L54, Y55, H117, F118, F129, T226, W227, N306 and Y310. Each residue in the cavity, except for the catalytic residues Y55 and H117, was systematically mutated to alanine to determine the role of the individual residues in steroid recognition. Binding data and kinetic parameters (K(d), k(cat), K(m) and k(cat)/K(m)) of the homogeneous mutants were compared with that of the wild type (WT) enzyme. Titration of the intrinsic tryptophan fluorescence with NADPH demonstrated that cofactor binding was unaltered. However, binding of the steroid hormones testosterone and progesterone to the E.NADPH binary complex was affected to varying degrees. The largest effects on K(d) were an 8-fold decrease in affinity for testosterone and a 50-fold decrease in affinity for progesterone. The mutants bound both hormones in the same rank-order except for W227A, where the binding of progesterone was more adversely affected. A series of 3alpha-hydroxysteroid substrates (A/B trans- and cis-ring fused C(19) and C(21) steroids) were used to determine the ability of each mutant to catalyze steroid turnover. The alanine mutants that retained k(cat)/K(m) values similar to WT were those in which alanine substituted short polar residues such as T24A and T226A. The mutants with the lowest catalytic efficiencies were those in which alanine substituted aromatic residues such as W227A and F129A. The loss in catalytic efficiency was due to large changes in k(cat) (up to 1000-fold), but not K(m). Molecular modeling of the alanine mutants showed that changes in the reaction trajectory defined by the angles and distances by groups that participate in catalysis correlate with changes in k(cat). These results highlight the importance of steroid binding site residues in dictating the proper orientation of substrates to achieve high catalytic turnover while having minimal effects on hormone affinity.

The aldo-keto reductase superfamily homepage.

The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates. A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit www.med.upenn.edu/akr). Investigators have been diligent in submitting sequences of functional proteins to the Web site. With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14). The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest. Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases. Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase). Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site. Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity. Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers. To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed. For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated. The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.