This article reports the results of a recent study to evaluate the usefulness of physical models of molecular structures as a new tool with which to teach concepts of molecular structure and function. Of seven different learning tools used by students in this introductory biochemistry class, the use of the physical models in a laboratory was rated as most useful. These results suggest that physical models can play an important role in capturing the interest of students in the subject of molecular structure and function. These physical models often stimulate more sophisticated questions in the minds of students, which can then be more appropriately explored using computer visualization tools.

The first detailed kinetic study of in vitro reactions between serum albumin and the secondgeneration gold drug 3,4,glucopytanosato-S-) gold(I)] and its tri-i-propylphosphine analogue, .iPr3PAuSATg, are reported. The reactions were investigated using Penefsky spun columns and NMR saturation transfer kinetics.Based on the Penefsky column data, the binding of the Et3PAuSATg to AIbSH (0.600 mM) was complete when gold concentrations were limiting: 0.093, 0.151, and 0.225 mM. The reaction is biphasic. The fast phase is defined by a first order rate constant, (k 2.94 _+ 0.24 x 10 -2 sec"1) and accounts for 95% of the Au(I) bound. This phase is first order with respect to albumin, and zero order with respect to auranofin. A minor, slower step (k 2 2.26 _+ 0.26 x 10 -3 sec"1), which accounts for only 5% of the reaction, is also first order with respect to albumin, and zero order with respect to auranofin. For .iPr3PAuSATg, only the first step was observed, k 1.4 +_ 0.1 x 10 -2 s "1 and is first order in albumin and independent of iPr3PAuSATg concentration. 31p.NMR saturation transfer experiments utilizing the auranofin analogue, .iPr3PAuSATg, under equilibrium conditions with excess .iPr3PAuSATg and ATgSH yielded second order rate constants for both the forward (1.0 x 102 M " sec"1) and the reverse (3.9 x 101 M "1 sec"1) directions. A multi-step mechanism involving a conformationally altered albumin species to which gold binds was developed using the steady-state approximation. The mechanism accounts for the different reaction orders observed under the two set of conditions. A rate determining conformational change on the albumin governs the reaction as monitored by the Penefsky columns. A second order reaction of R3PAuSATg at Cys-34 is observed under the NMR conditions. These results are the first quantitative determination of auranofin-albumin reaction kinetics and the first mechanistic study of the reaction.A novel binding mechanism, association of auranofin in the pocket of albumin-disulfide species can be detected by Penefsky and HummeI-Dreyer gel chromatographic techniques, but not by conventional gel-exclusion chromatography. This complex albumin-auranofin complex is weaklybound and readily dissociates.

The hereditary deficiency of 3-hydroxy-3-methylglutaryl (HMG) CoA lyase (HL; OMIM 246450 [http://www3.ncbi.nlm.nih. gov:80/htbin-post/Omim/dispmim?246450]) results in episodes of hypoketotic hypoglycemia and coma and is reported to be frequent and clinically severe in Saudi Arabia. We found genetic diversity among nine Saudi HL-deficient probands: six were homozygous for the missense mutation R41Q, and two were homozygous for the frameshift mutation F305fs(-2). In 32 non-Saudi HL-deficient probands, we found three R41Q alleles and also discovered four other deleterious point mutations in codons 41 and 42: R41X, D42E, D42G, and D42H. In purified mutant recombinant HL, all four missense mutations in codons 41 and 42 cause a marked decrease in HL activity. We developed a screening procedure for HL missense mutations that yields residual activity at levels comparable to those obtained using purified HL peptides. Codons 41 and 42 are important for normal HL catalysis and account for a disproportionate 21 (26%) of 82 of mutant alleles in our group of HL-deficient probands.

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is inactivated by diethyl pyrocarbonate (DEPC); activity can be fully restored by incubation with hydroxylamine. Protection against DEPC inactivation is afforded by a substrate analogue, suggesting an active site location for a DEPC target. Included in the inherited defects that map within the HMG-CoA lyase gene is a point mutation that results in an arginine substitution for histidine 233, one of only two invariant histidines. These observations prompted a functional test of the importance of His-233. The mutant lyases H233R, H233A, and H233D were overexpressed in Escherichia coli, isolated, and kinetically characterized. In H233D, DEPC targets one less histidine than was measured using wild-type lyase, supporting the assignment of wild-type lyase His-233 as one of the DEPC targets. Substitution of His-233 results in diminution of activity by ϳ4 orders of magnitude. K m values of the mutant lyases for both substrate HMG-CoA and activator divalent cation (Mg 2؉ or Mn 2؉) are comparable to the values measured for wild-type enzyme, indicating that these enzymes retain substantial structural integrity. This conclusion is reinforced by the observation that the affinity label, 2-butynoyl-CoA, stoichiometrically modifies the mutant lyases, indicating that they contain a full complement of active sites. In view of these data suggesting that the structures of these mutant lyases closely approximate that of the wild-type enzyme, their observed 10 4 -fold diminution in catalytic efficiency supports assignment to His-233 of a role in the chemistry of HMG-CoA cleavage.3-Hydroxy-3-methylglutaryl-CoA lyase (EC 4.1.3.4) catalyses the cleavage of HMG-CoA 1 into acetoacetate and acetylCoA. This reaction is an important step in both the ketogenic HMG-CoA cycle (1) and the leucine catabolic pathway (2). The chemistry of HMG-CoA cleavage (Equation 1) is believed to involve a retro-Claisen condensation in which both a general acid and base are required for catalysis.As depicted in Equation 1, a general base abstracts a proton from the C3 hydroxyl of HMG-CoA, which leads to ketone formation and cleavage of the C2-C3 bond. This generates a transient carbanionic form of acetyl-CoA, which is quenched by a proton provided by a general acid. Neither the general acid nor the base has been unequivocally assigned.Using affinity labeling techniques, a cysteine residue (Cys-237) was mapped to the active site of prokaryotic HMG-CoA lyase (3). Human HMG-CoA lyase has recently been expressed in Escherichia coli cells and purified to homogeneity (4). Using this expression system, the active site cysteine (Cys-266) of human HMG-CoA lyase was altered by site-directed mutagenesis to determine whether this amino acid was a key component of the catalytic apparatus (5). Upon conservative substitution with serine or alanine, a 10 3 -10 4 -fold decrease in the rate of catalysis and an altered pH/rate profile was observed (5). The mutagenesis data were in accord with the protein chemistry data suggesting that ...

The role of cysteine 266 in human 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase, a residue that is homologous to a cysteine mapped to the active site of prokaryotic HMG-CoA lyase by protein chemistry approaches, has been investigated by site-directed mutagenesis. Both the wild-type human enzyme and a C323S variant, in which a regulatory sulfhydryl has been eliminated without any negative effect on catalytic activity (Roberts, J. R., Narasimhan, C., Hruz, P. W., Mitchell, G. A., and Miziorko, H. M. (1994) J. Biol. Chem. 269, 17841-17846), were used as models. Mutant enzymes C266A, C266A/C323S, C266S, and C266S/C323S were overexpressed in Escherichia coli and purified to homogeneity. In all cases, kinetic characterization indicated that the Km value for HMG-CoA was not substantially different from the value measured using wild-type human lyase, suggesting that no serious structural perturbation occurs upon replacing Cys-266. A dissociable divalent cation (Mn2+ or Mg2+), which is required for activity in both native and C323S enzymes, is also an essential component for activity in each of the Cys-266 mutants. The structural integrity of the human mutants was further indicated by Mn2+ binding studies, which demonstrate similarities not only in the activator cation binding stoichiometries, but also in the KD values for Mn2+ as determined for wild-type and mutant C266A or C266S proteins. Purified C266A and C266A/C323S mutants both displayed approximately 1.3 x 10(4)-fold diminution in specific activity, while the kcat value was diminished in both C266S and C266S/C323S by approximately 9.9 x 10(2)-fold. This large diminution in catalytic efficiency in enzyme variants that display no substantial structural perturbations is in accord with an active-site assignment to Cys-266 and qualifies its sulfhydryl group for consideration as a component of the catalytic apparatus.