This review provides a description of the biochemistry and enzymology of the alpha-aminoadipate pathway for lysine biosynthesis in fungi. The alpha-aminoadipate pathway is unique to fungi and is thus a potential target for the rational design of antifungal drugs. The present state of knowledge of the mechanisms of the seven enzymes in the pathway is presented, as well as detailed information with respect to structures and mechanisms of homocitrate synthase, saccharopine reductase, and saccharopine dehydrogenase.

Homocitrate synthase (acetyl-coenzyme A: 2-ketoglutarate C-transferase; E.C. 2.3.3.14) (HCS) catalyzes the condensation of acetyl-CoA (AcCoA) and alpha-ketoglutarate (alpha-KG) to give homocitrate and CoA. Although the structure of an HCS has not been solved, the structure of isopropylmalate synthase (IPMS), a homologue, has been solved (Koon, N., Squire, C. J., and Baker, E. N. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 8295-8300). Three active site residues in IPMS, Glu-218, His-379, and Tyr-410, were proposed as candidates for catalytic residues involved in deprotonation of the methyl group of AcCoA prior to the Claisen condensation to give homocitrylCoA. All three of the active site residues in IPMS are conserved in the HCS from Saccharomyces cerevisiae. Site-directed mutagenesis has been carried out to probe the role of the homologous residues, Glu-155, His-309, and Tyr-320, in the S. cerevisiae HCS. No detectable activity was observed for the H309A and H309N mutant enzyme, but a slight increase in activity was observed for H309A in the presence of 300 mM imidazole, which is still 1000-fold lower than that of wild type (wt). The E155Q and E155A mutant enzymes exhibited 1000-fold lower activity than wt. The activity of E155A, but not of E155Q, could be partially rescued by formate; a K act of 60 mM with a modest 4-fold maximum activation was observed. In the presence of formate, E155A gives k cat, K AcCoA, and K alpha-KG values of 0.0031 s (-1), 13 muM, and 39 microM, respectively, while a primary kinetic deuterium isotope effect of about 1.4 was obtained on V, with deuterium in the methyl of AcCoA. The pH dependence of k cat for E155A in the presence of formate gave a p K a of 7.9 for a group that must be protonated for optimum activity, similar to that observed for the wt enzyme. However, a partial change was observed on the acid side of the profile, compared to the all or none change observed for wt giving a p K a of about 6.7. The k cat for E155Q decreased at high pH, similar to the wt enzyme, but was pH independent at low pH. The Y320F mutant enzyme only lost 25-fold activity compared to that of the wt, giving k cat, K AcCoA, and K alpha-KG values of 0.039 s (-1), 33 microM, and 140 microM, respectively, and a primary kinetic deuterium isotope effect of 1.3 and 1.8 on V/ K AcCoA and V, respectively; the pH dependence of k cat was similar to that of the wt. These data, combined with a constant pH molecular dynamics simulation study, suggest that a catalytic dyad comprising Glu-155 and His-309 acts to deprotonate the methyl group of AcCoA, while Tyr320 is likely not directly involved in catalysis, but may aid in orienting the reactant and/or the catalytic dyad.

Homocitrate synthase (acetyl-coenzyme A:2-ketoglutarate C-transferase; E.C. 2.3.3.14) catalyzes the condensation of AcCoA and alpha-ketoglutarate to give homocitrate and CoA. The enzyme was found to be a Zn-containing metalloenzyme using inductively coupled plasma mass spectrometry. Dead-end analogues of alpha-ketoglutarate were used to obtain information on the topography of the alpha-ketoglutarate binding site. The alpha-carboxylate and alpha-oxo groups of alpha-ketoglutarate are required for optimum binding to coordinate to the active site Zn. Optimum positioning of the alpha-carboxylate, alpha-oxo, and gamma-carboxylate of alpha-ketoglutarate is likely mimicked by the location in space of the 2-carboxylate, pyridine nitrogen, and 4 carboxylate of pyridine 2,4-dicarboxylate. The pH dependence of the kinetic parameters was determined to obtain information on the chemical mechanism of homocitrate synthase. The V profile is bell shaped with slopes of 1 and -1, giving pKa values of 6.7 and 8.0, while V/K(AcCoA) exhibits a slope of 2 on the acidic side with an average pKa value of 6.6 and a slope of -2 on basic side of the profile with an average pKa value of 8.2. The V/K(alpha-Kg) pH-rate profile exhibits a single pKa of 6.9 on the acidic side and two on the basic side with an average value of 7.8. The pH dependence of the Ki for glyoxylate, a competitive inhibitor vs alpha-ketoglutarate, gives a pKa of 7.1 for a group, required to be protonated for optimum binding. Data suggest a chemical mechanism for the enzyme in which alpha-ketoglutarate first binds to the active site Zn via its alpha-carboxylate and alpha-oxo groups, followed by acetyl-CoA. A general base then accepts a proton from the methyl of acetyl-CoA, and a general acid protonates the carbonyl of alpha-ketoglutarate in the formation of homocitryl-CoA. The general acid then acts as a base in deprotonating Zn-OH2 in the hydrolysis of homocitryl-CoA to give homocitrate and CoA. A solvent deuterium kinetic isotope effect of 1 is measured for homocitrate synthase, while a small pH-independent primary kinetic deuterium isotope effect (approximately 1.3) is observed using deuterioacetyl-CoA. Data suggest rate-limiting condensation to form the alkoxide of homocitryl-CoA, followed by hydrolysis to give products.

BACKGROUND Shoulder is the most injured part in table tennis players, and it takes multiple roles in transmitting power and striking the center of the ball during the stroke. Proprioception is strongly correlated with high level of athletic performance. It is customary to assume that there is a correlation between proprioception and muscle strength and therefore proprioceptive assessment and rehabilitation is often neglected. AIM To investigate the correlation between isokinetic muscle strength and proprioception in the internal and external rotation muscle groups of elite Chinese male table tennis players, to provide reference for physical training and rehabilitation of elite table tennis players. METHODS A total of 19 national elite table tennis players from the Chinese National Table Tennis Team were recruited in this research. All of them had more than 10 years training experience and had participated major competitions such as the National Games and World Youth Championships. IsoMed 2000 was used to test the peak torque of internal and external rotation isokinetic concentric contraction of the athletes' bilateral shoulder joints at low speed (60°/s) and high speed (180°/s) respectively; IsoMed 2000 was used to conduct the Joint Position Reproduction test to evaluate the athletes' proprioceptive ability capacity at low speed (60°/s) and high speed (180°/s) respectively. If the data satisfied the normal distribution, the correlation between the differences in peak torque s and angles in different directions was analyzed using a Pearson simple linear model; otherwise, Spearman correlation analysis was used. The comparison of proprioceptive ability between the table tennis racket-holding hand and non-racket-holding hands was performed using independent samples t -test if the data satisfied a normal distribution; otherwise, the Mann-Whitney U test was used. RESULTS There was no direct linear correlation between the strength and proprioceptive correlation analysis at slow speed (60°/s) and fast speed (180°/s) in the racket-holding hand; At the slow speed (60°/s) and fast speed (180°/s), there was no correlation between muscle strength and proprioception in the non-racket-holding hand except for the internal rotation variable error (VE) and external rotation relative peak torque, which showed a moderate positive correlation ( r = 0.477, P < 0.05), ( r = 0.554, P < 0.05). The internal rotation’s constant error (CE) and VE were 1.06 ± 3.99 and 2.94 ± 2.16, respectively, for the racket-holding hand, and -3.36 ± 2.39 and 1.22 ± 0.93, respectively, for the non-racket-holding hand; the internal rotation’s CE, VE of the racket-holding hand was lower than that of the non-racket-holding hand, and there was a highly significant difference ( P ...

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